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Multiple three-dimensional (3D) tumour organoid models assisted by multi-omics and Artificial Intelligence (AI) have contributed greatly to preclinical drug development and precision medicine. The intrinsic ability to maintain genetic and phenotypic heterogeneity of tumours allows for the reconciliation of shortcomings in traditional cancer models. While their utility in preclinical studies have been well established, little progress has been made in translational research and clinical trials. In this review, we identify the major bottlenecks preventing patient-derived tumour organoids (PDTOs) from being used in clinical setting. Unsuitable methods of tissue acquisition, disparities in establishment rates and a lengthy timeline are the limiting factors for use of PDTOs in clinical application. Potential strategies to overcome this include liquid biopsies via circulating tumour cells (CTCs), an automated organoid platform and optical metabolic imaging (OMI). These proposed solutions accelerate and optimize the workflow of a clinical organoid drug screening. As such, PDTOs have the potential for potential applications in clinical oncology to improve patient outcomes. If remarkable progress is made, cancer patients can finally benefit from this revolutionary technology.
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Preclinical models of diffuse-type gastric cancer (DGC) that reliably predict clinical activity of novel compounds are lacking. To overcome the problem of poor tumor cellularity in DGC, we used cells from malignant ascites to establish DGC patient-derived xenograft (PDX) models that recapitulate the primary cancer. Cells in PDX model GAGA6 with FGFR2 amplification were sensitive to AZD4547, a potent FGFR inhibitor that is being clinically evaluated for FGFR-aberrant cancer types. Intermittent in vivo treatment of GAGA6 tumors with AZD4547 gave rise to PDX tumors with acquired resistance to AZD4547, GAGA6-R. Surprisingly, there were no mutations in the FGFR2 gene in GAGA6-R, negating gatekeeper mutations as a mechanism of drug resistance. Phosphorylation of FGFR2 and downstream signaling molecules AKT/PKB and MAPK/ERK remained inhibited by AZD4547. Further analysis of signaling pathways identified AKT-independent phosphorylation and inhibition of GSK3ß as a mechanism of drug resistance in GAGA6-R cells. Treatment of GAGA6-R cells with protein kinase C (PKC) inhibitor H7 in combination with AZD4547 led to dephosphorylation and activation of GSK3ß with concomitant downregulation of MCL-1 and BCL-XL. Combined treatment with AZD4547 and H7 in vitro synergistically enhanced cell death in GAGA6-R but not GAGA6 cells. Furthermore, midostaurin, a multikinase inhibitor with PKC-inhibiting activity, in part reversed resistance of GAGA6-R tumor to AZD4547 in vivo Our results suggest that upon challenge with FGFR inhibitors, FGFR2-amplified tumors that are highly dependent on FGFR2 signaling for survival rapidly develop resistance by switching to a PKC-mediated inhibition of GSK3ß to gain a survival advantage. Mol Cancer Ther; 17(1); 232-42. ©2017 AACR.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias Gástricas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , TransfecçãoRESUMO
ASC amino acid transporter 2 (ASCT2), also known as solute linked carrier family 1 member A5 (SLC1A5) is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be up-regulated in a variety of cancerous tissues including stomach, liver, and kidney. In this study, we evaluated anti-tumor efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, which has a neutralizing activity against glutamine uptake, as a therapeutic antibody against gastric cancer and explored clinical predictive biomarker candidates by utilizing patient-derived xenograft (PDX) mouse models. Anti-tumor efficacy studies revealed that some of the PDX models used were responsive to KM8094 and the others were not. Interestingly, we observed a correlation between anti-tumor efficacy and low antigen expression as well as low basal levels of glutamine uptake, suggesting ASCT2 expression level could be a potential predictive biomarker for KM8094. We then further explored predictive biomarker candidates by multi-omics analysis on gastric cancer PDX mouse models. As a result, a few potential candidates such as TFF2, MUC13, and ANG were selected by gene expression and DNA methylation array analyses. In addition, metabolomics analysis revealed clear differences in intracellular energy status and redox status between responsive and non-responsive PDX models.
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ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell viability and that BCL6 overexpression is associated with worse prognosis. In a somatic transgenic mouse model, depletion of Bcl6 inhibits the progression of KrasG12V-driven high-grade glioma. Transcriptome analysis demonstrates the involvement of BCL6 in tumor protein p53 (TP53), erythroblastic leukemia viral oncogene homolog (ErbB), and MAPK signaling pathways. Indeed, BCL6 represses the expression of wild-type p53 and its target genes in GBM cells. Knockdown of BCL6 augments the activation of TP53 pathway in response to radiation. Importantly, we discover that receptor tyrosine kinase AXL is a transcriptional target of BCL6 in GBM and mediates partially the regulatory effects of BCL6 on both MEK-ERK (mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase) and S6K-RPS6 (ribosomal protein S6 kinase-ribosomal protein S6) axes. Similar to BCL6 silencing, depletion of AXL profoundly attenuates GBM proliferation both in vitro and in vivo. Moreover, targeted inhibition of BCL6/nuclear receptor corepressor 1 (NCoR) complex by peptidomimetic inhibitor not only significantly decreases AXL expression and the activity of MEK-ERK and S6K-RPS6 cascades but also displays a potent antiproliferative effect against GBM cells. Together, these findings uncover a glioma-promoting role of BCL6 and provide the rationale of targeting BCL6 as a potential therapeutic approach.
Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Gefitinibe , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos Mutantes , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/genética , Quinazolinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
RUNX3 functions as a tumor suppressor in the gastric epithelium, where its inactivation is frequently observed during carcinogenesis. We identified IL23A as a RUNX3 target gene in gastric epithelial cells. This was confirmed in a series of in vitro analyses in gastric epithelial cell lines. In elucidating the underlying regulatory network, we uncovered a prominent role for the TNF-α/NF-κB pathway in activating IL23A transcription. Moreover, the activating effect of TNF-α was markedly augmented by the infection of Helicobacter pylori, the primary cause of human gastritis. Of note, H. pylori utilized the CagA/SHP2 pathway to activate IL23A, as well as the induction of the NOD1 pathway by iE-DAP. Importantly, RUNX3 synergized strongly with these physiologically relevant stimuli to induce IL23A. Lastly, we present evidence for the secretion of IL23A by gastric epithelial cells in a form that is distinct from canonical IL-23 (IL23A/IL12B).
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Mucosa Gástrica/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Ativação Transcricional , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Mucosa Gástrica/microbiologia , Helicobacter pylori/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Subunidade p19 da Interleucina-23/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.
Assuntos
Hipóxia Celular/genética , Antígenos de Histocompatibilidade/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco/metabolismo , Neoplasias Testiculares/patologia , Animais , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Análise Serial de TecidosRESUMO
The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers.
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Biomarcadores Tumorais/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/patologia , Animais , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Molécula de Adesão da Célula Epitelial , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Neoplasias Gástricas/metabolismo , Células Tumorais CultivadasRESUMO
The transcription factor RUNX3 functions as a tumor suppressor in the gastrointestinal epithelium, where its loss is an early event in carcinogenesis. While RUNX3 acts concurrently as a mediator of TGF-ß signaling and an antagonist of Wnt, the cellular changes that follow its loss and their contribution to tumorigenicity are not fully understood. Here, we report that the loss of Runx3 in gastric epithelial cells results in spontaneous epithelial-mesenchymal transition (EMT). This produces a tumorigenic stem cell-like subpopulation, which remarkably expresses the gastric stem cell marker Lgr5. This phenomenon is due to the compounding effects of the dysregulation of the TGF-ß and Wnt pathways. Specifically, Runx3(-/-) p53(-/-) gastric epithelial cells were unexpectedly sensitized for TGF-ß-induced EMT, during which the resultant induction of Lgr5 was enhanced by an aberrantly activated Wnt pathway. These data demonstrate a protective role for RUNX3 in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness.
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Transformação Celular Neoplásica/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Neoplasias Gastrointestinais/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/patologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Western Blotting , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação para Baixo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso/farmacologia , Inibidores de Proteassoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Quinase C Ativada , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Valproic acid (VPA) is a histone deacetylase inhibitor previously shown to promote the proliferation and self-renewal of CD34(+) hematopoietic cells. We tested the effect of VPA in conjunction with the selective amplification technology developed by Viacell Inc. Stem cells enriched from frozen cord blood were cultured for 7 d, subjected to reselection and grown in fresh medium for a further 7 d. Treatment with VPA resulted in an average two-fold higher expansion of CD45(+)34(+) cells compared with control. Furthermore, VPA-treatment induced higher numbers of CD45(+)34(+) cells to reside in the S phase than control cultured cells and resulted in a 2.5-fold upregulation in HOXB4 expression. Importantly, VPA-treated cells reconstituted hematopoiesis in non-obese diabetic/severe combined immunodeficient mice with a six-fold higher efficiency than control cells. Collectively, our results indicate that VPA, already used clinically for neurologic disorder treatment, is a useful additive for the ex vivo culture of hematopoietic stem/progenitor cells to enhance engraftment efficiency.
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Sangue Fetal , Células-Tronco Hematopoéticas/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Antígenos CD34/imunologia , Sequência de Bases , Ciclo Celular , Meios de Cultura Livres de Soro , Primers do DNA , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Inibidores de Histona Desacetilases , Humanos , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.
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Apoptose/efeitos dos fármacos , Benzofenantridinas/farmacologia , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Caspases/metabolismo , Células Cultivadas , Ciclosporina/farmacologia , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Sensibilidade e EspecificidadeRESUMO
Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently, a new pluripotent population of umbilical cord blood (UCB) cells, unrestricted somatic stem cells (USSCs), with intrinsic and directable potential to develop into mesodermal, endodermal, and ectodermal fates, has been identified. In this study, we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold, respectively; p < .05), with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells, as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation, with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells, generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p < .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients.
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Antígenos CD34/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/citologia , Sangue Fetal/transplante , Células-Tronco/citologia , Animais , Células da Medula Óssea/citologia , Separação Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.
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Alcaloides/química , Benzofenantridinas/química , Isoquinolinas/química , Modelos Moleculares , Proteína bcl-X/química , Alcaloides/farmacologia , Motivos de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Benzofenantridinas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Mapeamento de Epitopos , Humanos , Isoquinolinas/farmacologia , Conformação Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismoRESUMO
Apoptotic stimuli induce conformational changes in Bax and trigger its translocation from cytosol to mitochondria. Upon assembling into the mitochondrial membrane, Bax initiates a death program through a series of events, culminating in the release of apoptogenic factors such as cytochrome c. Although it is known that Bax is one of the key factors for integrating multiple death signals, the mechanism by which Bax functions in mitochondria remains controversial. We have previously identified modulator of apoptosis-1 (MAP-1) as a Bax-associating protein, but its functional relationship with Bax in contributing to apoptosis regulation remains to be established. In this study, we show that MAP-1 is a critical mitochondrial effector of Bax. MAP-1 is a mitochondria-enriched protein that associates with Bax only upon apoptotic induction, which coincides with the release of cytochrome c from mitochondria. Small interfering RNAs that diminish MAP-1 levels in mammalian cell lines confer selective inhibition of Bax-mediated apoptosis. Mammalian cells with stable expression of MAP-1 small interfering RNAs are resistant to multiple apoptotic stimuli in triggering apoptotic death as well as in inducing conformation change and translocation of Bax. Similar to Bax-deficient cells, MAP-1-deficient cells exhibit aggressive anchorage-independent growth. Remarkably, recombinant Bax- or tBid-mediated release of cytochrome c from isolated mitochondria is significantly compromised in the MAP-1 knockdown cells. We propose that MAP-1 is a direct mitochondrial target of Bax.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologia , Proteína X Associada a bcl-2/metabolismo , Animais , Fracionamento Celular , Citocromos c/metabolismo , Fibroblastos , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Imunoprecipitação , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
1. Proteins of the Bcl-2 family are central regulators of apoptosis and are thought to act primarily on the mitochondria. 2. Members of the Bcl-2 family possess either anti-apoptotic or pro-apoptotic function. They are characterized by the presence of conserved sequence motifs, known as Bcl-2 homology (BH) domains. Anti-apoptotic members share all four BH domains, designated as BH1-4; the multidomain pro-apoptotic members contain BH1-3 domains, whereas another subgroup of pro-apoptotic members only have a BH3 domain. 3. The BH3-only proteins act as sensors for distinct apoptosis pathways, whereas multidomain pro-apoptotic Bax and Bak are executioners of death orders relayed by the BH3-only proteins. 4. Anti-apoptotic Bcl-2 family members appear to function, at least in part, by interacting with and antagonizing pro-apoptotic family members. The BH1-3 domains of BclXL form an elongated hydrophobic groove, which is the docking site for the BH3 domains of pro-apoptotic binding partners. 5. The deregulation of the various Bcl-2 proteins has been implicated in many pathological conditions. 6. Knowledge derived from the understanding of the function and regulation of the Bcl-2 family of proteins has allowed us to contemplate new therapeutic strategies for diseases where apoptosis signalling mechanisms can potentially be manipulated. 7. The anti-apoptotic Bcl-2 members have been targeted successfully using an antisense approach, BH3-peptides and small molecular weight chemicals that are inhibitors of their anti-apoptotic function.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Oligonucleotídeos Antissenso/uso terapêutico , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Transdução de SinaisRESUMO
The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.