RESUMO
BACKGROUND: PM2.5, a known public health risk, is increasingly linked to intestinal disorders, however, the mechanisms of its impact are not fully understood. PURPOSE: This study aimed to explore the impact of chronic PM2.5 exposure on intestinal barrier integrity and to uncover the underlying molecular mechanisms. METHODS: C57BL/6 J mice were exposed to either concentrated ambient PM2.5 (CPM) or filtered air (FA) for six months to simulate urban pollution conditions. We evaluated intestinal barrier damage, microbial shifts, and metabolic changes through histopathology, metagenomics, and metabolomics. Analysis of the TLR signaling pathway was also conducted. RESULTS: The mean concentration of PM2.5 in the CPM exposure chamber was consistently measured at 70.9 ± 26.8 µg/m³ throughout the study period. Our findings show that chronic CPM exposure significantly compromises intestinal barrier integrity, as indicated by reduced expression of the key tight junction proteins Occludin and Tjp1/Zo-1. Metagenomic sequencing revealed significant shifts in the microbial landscape, identifying 35 differentially abundant species. Notably, there was an increase in pro-inflammatory nongastric Helicobacter species and a decrease in beneficial bacteria, such as Lactobacillus intestinalis, Lactobacillus sp. ASF360, and Eubacterium rectale. Metabolomic analysis further identified 26 significantly altered metabolites commonly associated with intestinal diseases. A strong correlation between altered bacterial species and metabolites was also observed. For example, 4 Helicobacter species all showed positive correlations with 13 metabolites, including Lactate, Bile acids, Pyruvate and Glutamate. Additionally, increased expression levels of TLR2, TLR5, Myd88, and NLRP3 proteins were noted, and their expression patterns showed a strong correlation, suggesting a possible involvement of the TLR2/5-MyD88-NLRP3 signaling pathway. CONCLUSIONS: Chronic CPM exposure induces intestinal barrier dysfunction, microbial dysbiosis, metabolic imbalance, and activation of the TLR2/5-MyD88-NLRP3 inflammasome. These findings highlight the urgent need for intervention strategies to mitigate the detrimental effects of air pollution on intestinal health and identify potential therapeutic targets.
Assuntos
Disbiose , Inflamassomos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Proteína 3 que Contém Domínio de Pirina da Família NLR , Material Particulado , Receptor 2 Toll-Like , Receptor 5 Toll-Like , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Disbiose/induzido quimicamente , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Material Particulado/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptor 2 Toll-Like/metabolismo , Camundongos , Receptor 5 Toll-Like/metabolismo , Poluentes Atmosféricos/toxicidade , Masculino , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/microbiologiaRESUMO
Triple-negative breast cancer is an aggressive subtype of breast cancer that is primarily treated using systemic chemotherapy due to the lack of a specific cell surface marker for drug delivery. Cancer cell-specific aptamer-mediated drug delivery is a promising targeted chemotherapy for marker-unknown cancers. Using a poorly differentiated carcinoma cell-specific DNA aptamer (PDGC21T), we formed a self-assembling circinate DNA nanoparticle (Apt21TNP) that binds triple-negative breast cancer cells. Using our previously designed pH-sensitive dendrimer-conjugated doxorubicin (DDOX) as the payload, we found that each nanoparticle loaded 30 doxorubicin molecules to form an Apt21TNP-DDOX nanomedicine that is stable in human plasma. Upon cell binding, Apt21TNP-DDOX is internalized by triple-negative breast cancer cells through the macropinocytosis pathway. Once inside cells, the low pH microenvironment in lysosomes induces doxorubicin drug payload release from Apt21TNP-DDOX. Our in vitro studies demonstrate that Apt21TNP-DDOX can preferentially bind triple-negative breast cancer cells to induce cell death. Furthermore, we show that Apt21TNP-DDOX can accumulate in subcutaneous MDA-MB-231 tumors in mice following systemic administration to reduce tumor burden, minimize side effects, and improve animal survival. Together, our results demonstrate that Apt21TNP-mediated doxorubicin delivery is a potent, targeted chemotherapy for triple-negative breast cancer that may alleviate side effects in patients.
Assuntos
Aptâmeros de Nucleotídeos , Nanopartículas , Nanoestruturas , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Doxorrubicina , Humanos , Camundongos , Nanopartículas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Microambiente TumoralRESUMO
The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7â nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5â nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Antivirais/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , COVID-19/metabolismo , Células HEK293 , Humanos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas de Cultura de Células , Mycoplasma/isolamento & purificação , Ligação Competitiva , Linhagem Celular Tumoral , Meios de Cultura , Contaminação por DNA , Citometria de Fluxo , Humanos , Mycoplasma/genéticaRESUMO
High-risk neuroblastoma often develops resistance to high-dose chemotherapy. The mTOR signaling cascade is frequently deregulated in human cancers and targeting mTOR signaling sensitizes many cancer types to chemotherapy. Here, using a panel of neuroblastoma cell lines, we found that the mTOR inhibitor INK128 showed inhibitory effects on both anchorage-dependent and independent growth of neuroblastoma cells and significantly enhanced the cytotoxic effects of doxorubicin (Dox) on these cell lines. Treatment of neuroblastoma cells with INK128 blocked the activation of downstream mTOR signaling and enhanced Dox-induced apoptosis. Moreover, INK128 was able to overcome the established chemoresistance in the LA-N-6 cell line. Using an orthotopic neuroblastoma mouse model, we found that INK128 significantly inhibited tumor growth in vivo. In conclusion, we have shown that INK128-mediated mTOR inhibition possessed substantial antitumor activity and could significantly increase the sensitivity of neuroblastoma cells to Dox therapy. Taken together, our results indicate that using INK128 can provide additional efficacy to current chemotherapeutic regimens and represent a new paradigm in restoring drug sensitivity in neuroblastoma.
Assuntos
Benzoxazóis/farmacologia , Complexos Multiproteicos/metabolismo , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.
Assuntos
Leucoceratose da Mucosa Hereditária/diagnóstico , Leucoceratose da Mucosa Hereditária/tratamento farmacológico , Animais , Humanos , Leucoceratose da Mucosa Hereditária/genética , Leucoceratose da Mucosa Hereditária/patologia , Mucosa Bucal/patologia , Doenças Raras/diagnóstico , Doenças Raras/tratamento farmacológico , Doenças Raras/genética , Doenças Raras/patologiaRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer comprised of cells that lack expression of targetable biomarkers. Nucleic acid aptamers are a group of molecular ligands that can specifically bind to their targets with high affinity. The ssDNA aptamer PDGC21-T recognizes poorly differentiated cancer cells and tumor tissues through an unidentified cell surface target(s). Because TNBC tumor cells are poorly differentiated, the aptamer PDGC21-T is a promising therapeutic candidate to target TNBC tumor cells. In vitro study revealed that synthetic aptamer probes selectively targeted TNBC cell lines. To assess aptamer immunotherapeutic targeting capability, we generated aptamer-engineered NK cells (ApEn-NK) using aptamer probes as a targeting ligand and NK cells as a therapeutic agent. Cell clustering formation assays revealed that ApEn-NK bound both suspended and adherent TNBC cells with high affinity. In a functional study, ApEn-NK treatment triggered apoptosis and death of cultured TNBC cells. Finally, systemic administration of ApEn-NK in mice harboring TNBC xenografts resulted in significant inhibition of lung metastasis relative to parental NK cell treatments. Unlike chemotherapy, ApEn-NK treatment did not affect body weight in treated mice. We demonstrate a novel approach for targeted TNBC immunotherapy.
Assuntos
Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Humanos , Imunoterapia , Células Matadoras Naturais/metabolismo , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Although targeted cancer therapy can induce higher therapeutic efficacy and cause fewer side effects in patients, the lack of targetable biomarkers on triple-negative breast cancer (TNBC) cells limits the development of targeted therapies by antibody technology. Therefore, we investigated an alternative approach to target TNBC by using the PDGC21T aptamer, which selectively binds to poorly differentiated carcinoma cells and tumor tissues, although the cellular target is still unknown. We found that synthetic aptamer probes specifically bound cultured TNBC cells in vitro and selectively targeted TNBC xenografts in vivo. Subsequently, to identify the target molecule on TNBC cells, we performed aptamer-mediated immunoprecipitation in lysed cell membranes followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Sequencing analysis revealed a highly conserved peptide sequence consistent with the cell surface protein CD49c (integrin α3). For target validation, we stained cultured TNBC and non-TNBC cells with an aptamer probe or a CD49c antibody and found similar cell staining patterns. Finally, competition cell-binding assays using both aptamer and anti-CD49c antibody revealed that CD49c is the biomarker targeted by the PDGC21T aptamer on TNBC cells. Our findings provide a molecular foundation for the development of targeted TNBC therapy using the PDGC21T aptamer as a targeting ligand.
RESUMO
Gemcitabine is a chemotherapeutic used clinically to treat a variety of cancers. However, because it lacks tumor cell specificity, gemcitabine may cause off-target cytotoxicity and adversely impact patients. To impart cancer cell specificity to gemcitabine and improve its therapeutic efficacy, we synthesized a unique aptamer-drug conjugate that carries a high gemcitabine payload (three molecules) via a dendrimer structure and enzymatically cleavable linkers for controlled intracellular drug release. First, linker-gemcitabinedendrimer-linker-gemcitabine products were produced, which had significantly lower cytotoxicity than an equimolar amount of free drug. Biochemical analysis revealed that lysosomal cathepsin B protease rapidly cleaved the dendritic linkers and released the conjugated gemcitabine as a free drug. Subsequently, the dendrimer-linker-gemcitabine was coupled with a cell-specific aptamer to form aptamer-gemcitabine conjugates. Functional assays confirmed that, under aptamer guidance, aptamer-gemcitabine conjugates were selectively bound to and then internalized by triple-negative breast cancer cells. Cellular therapy studies indicated that the aptamer-gemcitabine conjugates potentiated cytotoxic activity to targeted cancer cells but did not affect off-target control cells. Our study demonstrates a novel approach to aptamer-mediated targeted drug delivery that combines a high drug payload and an enzymatically controlled drug release switch to achieve higher therapeutic efficacy and fewer off-target effects relative to free-drug chemotherapy.
RESUMO
The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7â nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5â nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.
RESUMO
Hepatoblastoma (HB) is the most common pediatric liver malignancy. High-risk patients have poor survival, and current chemotherapies are associated with significant toxicities. Targeted therapies are needed to improve outcomes and patient quality of life. Most HB cases are TP53 wild-type; therefore, we hypothesized that targeting the p53 regulator Murine double minute 4 (MDM4) to reactivate p53 signaling may show efficacy. MDM4 expression was elevated in HB patient samples, and increased expression was strongly correlated with decreased expression of p53 target genes. Treatment with NSC207895 (XI-006), which inhibits MDM4 expression, or ATSP-7041, a stapled peptide dual inhibitor of MDM2 and MDM4, showed significant cytotoxic and antiproliferative effects in HB cells. Similar phenotypes were seen with short hairpin RNA (shRNA)-mediated inhibition of MDM4. Both NSC207895 and ATSP-7041 caused significant upregulation of p53 targets in HB cells. Knocking-down TP53 with shRNA or overexpressing MDM4 led to resistance to NSC207895-mediated cytotoxicity, suggesting that this phenotype is dependent on the MDM4-p53 axis. MDM4 inhibition also showed efficacy in a murine model of HB with significantly decreased tumor weight and increased apoptosis observed in the treatment group. This study demonstrates that inhibition of MDM4 is efficacious in HB by upregulating p53 tumor suppressor signaling.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Pré-Escolar , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Oxidiazóis/uso terapêutico , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neuroblastoma/tratamento farmacológico , Piperidonas/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Intervalo Livre de Doença , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Etoposídeo/uso terapêutico , Feminino , Seguimentos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/patologia , Piperidonas/uso terapêutico , Desdobramento de Proteína/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Camundongos Nus , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/patologia , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Maternal embryonic leucine zipper kinase (MELK) is known to modulate intracellular signaling and control cellular processes. However, the role of MELK in oncogenesis is not well defined. In this study, using two microarray datasets of neuroblastoma (NB) patients, we identified that MELK expression is significantly correlated to poor overall survival, unfavorable prognosis, and high-risk status. We found that MELK is a direct transcription target of MYCN and MYC in NB, and MYCN increases MELK expression via direct promoter binding. Interestingly, knockdown of MELK expression significantly reduced the phosphorylation of target protein Retinoblastoma (pRb) and inhibited NB cell growth. Furthermore, pharmacological inhibition of MELK activity by small-molecule inhibitor OTSSP167 significantly inhibited cell proliferation, anchorage-independent colony formation, blocked cell cycle progression, and induced apoptosis in different NB cell lines including a drug-resistant cell line. Additionally, OTSSP167 suppressed NB tumor growth in an orthotopic xenograft mouse model. Overall, our data suggest that MELK is a novel therapeutic target for NB and its inhibitor OTSSP167 is a promising drug for further clinical development.
RESUMO
Neuroblastoma (NB) is one of the most common pediatric malignancies in children. Abnormal activation of receptor tyrosine kinases contributes to the pathological development of NB. Therefore, targeting tyrosine kinase receptors to cure NB is a promising strategy. Here, we report that a multi-targeted tyrosine kinase inhibitor ponatinib inhibited NB cell proliferation and induced NB cell apoptosis in a dose-dependent manner. In addition, ponatinib suppressed the colony formation ability of NB cells. Mechanistically, ponatinib effectively inhibited the FGFR1-activated signaling pathway. Ponatinib also enhanced the cytotoxic effects of doxorubicin on NB cells. Furthermore, ponatinib demonstrated anti-tumor efficacy in vivo by inhibiting tumor growth in an orthotopic xenograft NB mouse model. In summary, our results showed that ponatinib inhibited NB growth both in vitro and in vivo.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Imidazóis/administração & dosagem , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Piridazinas/administração & dosagem , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Camundongos , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.
Assuntos
Fator de Crescimento Epidérmico/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Quinazolinas/farmacologia , Afatinib , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/biossíntese , Feminino , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Quinazolinas/administração & dosagem , Radiossensibilizantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in children. Aberrant activation of the non-receptor tyrosine kinases Src and c-Abl contributes to the progression of NB. Thus, targeting these kinases could be a promising strategy for NB therapy. In this paper, we report that the potent dual Src/Abl inhibitor bosutinib exerts anti-tumor effects on NB. Bosutinib inhibited NB cell proliferation in a dose-dependent manner and suppressed colony formation ability of NB cells. Mechanistically, bosutinib effectively decreased the activity of Src/Abl and PI3K/AKT/mTOR, MAPK/ERK, and JAK/STAT3 signaling pathways. In addition, bosutinib enhanced doxorubicin (Dox)- and etoposide (VP-16)-induced cytotoxicity in NB cells. Furthermore, bosutinib demonstrated anti-tumor efficacy in an orthotopic xenograft NB mouse model in a similar mechanism as of that in vitro. In summary, our results reveal that Src and c-Abl are potential therapeutic targets in NB and that the novel Src/Abl inhibitor bosutinib alone or in combination with other chemotherapeutic agents may be a valuable therapeutic option for NB patients.
Assuntos
Compostos de Anilina/farmacologia , Neuroblastoma/tratamento farmacológico , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Quinolinas/farmacologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Criança , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidoresRESUMO
Currently, preclinical testing of therapies for hepatoblastoma (HB) is limited to subcutaneous and intrasplenic xenograft models that do not recapitulate the hepatic tumors seen in patients. We hypothesized that injection of HB cell lines into the livers of mice would result in liver tumors that resemble their clinical counterparts. HepG2 and Huh-6 HB cell lines were injected, and tumor growth was monitored with bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Levels of human α-fetoprotein (AFP) were monitored in the serum of animals. Immunohistochemical and gene expression analyses were also completed on xenograft tumor samples. BLI signal indicative of tumor growth was seen in 55% of HepG2- and Huh-6-injected animals after a period of four to seven weeks. Increased AFP levels correlated with tumor growth. MRI showed large intrahepatic tumors with active neovascularization. HepG2 and Huh-6 xenografts showed expression of ß-catenin, AFP, and Glypican-3 (GPC3). HepG2 samples displayed a consistent gene expression profile most similar to human HB tumors. Intrahepatic injection of HB cell lines leads to liver tumors in mice with growth patterns and biologic, histologic, and genetic features similar to human HB tumors. This orthotopic xenograft mouse model will enable clinically relevant testing of novel agents for HB.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas Experimentais , Transplante de Neoplasias , Neovascularização Patológica , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Hep G2 , Xenoenxertos , Humanos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Treatment failure in high risk neuroblastoma (NB) is largely due to the development of chemotherapy resistance. We analyzed the gene expression changes associated with exposure to chemotherapy in six high risk NB tumors with the aid of the Connectivity Map bioinformatics platform. Ten therapeutic agents were predicted to have a high probability of reversing the transcriptome changes associated with neoadjuvant chemotherapy treatment. Among these agents, initial screening showed the EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279, had obvious cytotoxic effects on NB cell lines. Using a panel of NB cell lines, including MYCN nonamplified (SK-N-AS, SH-SY5Y, and CHLA-255), and MYCN amplified (NB-19, NGP, and IMR-32) cell lines, we found that YK-4-279 had cytotoxic effects on all lines tested. In addition, YK-4-279 also inhibited cell proliferation and anchorage-independent growth and induced cell apoptosis of these cells. YK-4-279 enhanced the cytotoxic effect of doxorubicin (Dox). Moreover, YK-4-279 was able to overcome the established chemoresistance of LA-N-6 NB cells. In an orthotopic xenograft NB mouse model, YK-4-279 inhibited NB tumor growth and induced apoptosis in tumor cells through PARP and Caspase 3 cleavage in vivo. While EWS-FLI1 fusion protein is not frequently found in NB, using the R2 public database of neuroblastoma outcome and gene expression, we found that high expression of EWSR1 was associated with poor patient outcome. Knockdown of EWSR1 inhibited the oncogenic potential of neuroblastoma cell lines. Taken together, our results indicate that YK-4-279 might be a promising agent for treatment of NB that merits further exploration.
RESUMO
Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo. We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.