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Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.
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Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Receptores ErbB , Gefitinibe , Neoplasias Pulmonares , Inibidores de Proteínas Quinases , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Gefitinibe/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Adrenergic ß2-agonists represent a mainstay in asthma management. Their chronic use has been associated with decreased bronchoprotection and rebound hyperresponsiveness. Here we investigate on the possible therapeutic advantage of a pharmacological association of ß2-agonists with montelukast, a highly selective leukotriene receptor antagonist, in modulating bronchial reactivity and controlling asthma features. The study has been conducted in vitro and in vivo and also takes advantage of the synthesis of a salt that gave us the possibility to simultaneously administer in vivo formoterol and montelukast (MFS). In vitro studies demonstrate that montelukast (1) preserves ß2-agonist response in isolated bronchi by preventing homologous ß2-adrenoceptor desensitization; (2) reduces desensitization by modulating ß2-receptor translocation in bronchial epithelial cells. In vivo studies demonstrate that sensitized mice receiving formoterol or montelukast display a significant reduction in airway hyperresponsiveness, but the ß2-agonist relaxing response is still impaired. Allergen challenge causes ß2 heterologous desensitization that is further increased by treatment in vivo with formoterol. Conversely MFS not only inhibits airway hyperresponsiveness but it rescues the ß2-agonist response. Histological analysis confirms the functional data, demonstrating an enhanced therapeutic efficiency of MSF in controlling also pulmonary metaplasia and lung inflammation. MFS is efficacious also when sensitized mice received the drug by local administration. In conclusion, the data obtained evidenced a therapeutic advantage in the association of ß2-agonists with montelukast in the control of asthma-like features and a better rescue bronchodilation response to ß2-agonists.
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Agonistas Adrenérgicos beta , Asma , Camundongos , Animais , Fumarato de Formoterol/farmacologia , Fumarato de Formoterol/uso terapêutico , Agonistas Adrenérgicos beta/uso terapêutico , Asma/tratamento farmacológico , Acetatos/farmacologia , Acetatos/uso terapêuticoRESUMO
Selection of cancer patients for treatment with immune checkpoint inhibitors remains a challenge due to tumour heterogeneity and variable biomarker detection. PD-L1 expression in 24 surgical chordoma specimen was determined immunohistochemically with antibodies 28-8 and E1L3N. The ability of patient-derived organoids to detect treatment effects of nivolumab was explored by quantitative and qualitative immunofluorescence and FACS analysis. The more sensitive antibody, E1L3N (ROC = 0.896, p = 0.001), was associated with greater tumour diameters (p = 0.014) and detected both tumour cells and infiltrating lymphocytes in 54% of patients, but only 1-15% of their cells. Organoids generated from PD-L1-positive patients contained both tumour cells and PD-1/CD8-positive lymphocytes and responded to nivolumab treatment with marked dose-dependent diameter reductions of up to 50% and increased cell death in both PD-L1-positive and negative organoids. Patient-derived organoids may be valuable to predict individual responses to immunotherapy even in patients with low or no immunohistochemical PD-L1 expression.
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Antígeno B7-H1/antagonistas & inibidores , Cordoma/metabolismo , Descoberta de Drogas/métodos , Imunoterapia/métodos , Nivolumabe/farmacologia , Organoides/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Cordoma/patologia , Cordoma/cirurgia , Feminino , Humanos , Imuno-Histoquímica/métodos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, ß1, ß3 and ß5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5ß1, αvß3 and αvß5 enhance cell adhesion to NETs, whereas low expression of α5ß1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.
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Adesão Celular/genética , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/genética , Transporte Proteico/genética , Membrana Celular , Armadilhas Extracelulares , Fibronectinas/genética , Citometria de Fluxo , Humanos , Neutrófilos/citologia , Neutrófilos/metabolismo , Células PC-3 , Peptídeos/genética , Ligação ProteicaRESUMO
The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.
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Antineoplásicos/isolamento & purificação , Poríferos/química , Tiazóis/isolamento & purificação , Valina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Humanos , Células MCF-7/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Tiazóis/química , Tiazóis/farmacologia , Valina/química , Valina/isolamento & purificação , Valina/farmacologiaRESUMO
Introduction: Diffuse pleural mesothelioma (DPM) of the pleura is a highly aggressive and treatment-resistant cancer linked to asbestos exposure. Despite multimodal treatment, the prognosis for DPM patients remains very poor, with an average survival of 2 years from diagnosis. Cisplatin, a platinum-based chemotherapy drug, is commonly used in the treatment of DPM. However, the development of resistance to cisplatin significantly limits its effectiveness, highlighting the urgent need for alternative therapeutic strategies. New selective inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have shown promise in various malignancies by inhibiting cell cycle progression and suppressing tumor growth. Recent studies have indicated the potential of abemaciclib for DPM therapy, and a phase II clinical trial has shown preliminary encouraging results. Methods: Here, we tested abemaciclib, palbociclib, and ribociclib on a panel of DPM cell lines and non-tumor mesothelial(MET-5A) cells. Results: Specifically, we focused on abemaciclib, which was the mosteffective cytotoxic agent on all the DPM cell lines tested. Abemaciclib reduced DPM cell viability, clonogenic potential, and ability to grow as three-dimensional (3D) spheroids. In addition, abemaciclib induced prolonged effects, thereby impairing second-generation sphere formation and inducing G0/G1 arrest and apoptosis/ necrosis. Interestingly, single silencing of RB family members did not impair cell response to abemaciclib, suggesting that they likely complement each other in triggering abemaciclib's cytostatic effect. Interestingly, abemaciclib reduced the phosphorylation of AKT, which is hyperactive in DPM and synergized with the pharmacological AKT inhibitor (AKTi VIII). Abemaciclib also synergized with cisplatin and reduced the viability of DPM cells with acquired resistance to cisplatin. Discussion: Overall, our results suggest that CDK4/6 inhibitors alone or in combination with standard of care should be further explored for DPM therapy.
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Background: Circulating Tumor Cells (CTCs) represent a small, heterogeneous population that comprise the minority of cells able to develop metastasis. To trap and characterize CTCs with metastatic attitude, a CXCL12-loaded hyaluronic-gel (CLG) was developed. CXCR4+cells with invasive capability would infiltrate CLG. Methods: Human colon, renal, lung and ovarian cancer cells (HT29, A498, H460 and OVCAR8 respectively) were seeded on 150 µl Empty Gels (EG) or 300 ng/ml CXCL12 loaded gel (CLG) and allowed to infiltrate for 16 h. Gels were then digested and fixed with 2 % FA-HAse for human cancer cell enumeration or digested with HAse and cancer cells recovered. CLG-recovered cells migrated toward CXCL12 and were tested for colonies/spheres formation. Moreover, CXCR4, E-Cadherin and Vimentin expression was assessed through flow cytometry and RT-PCR. The clinical trial "TRAP4MET" recruited 48 metastatic/advanced cancer patients (8 OC, 8 LC, 8 GBM, 8 EC, 8 RCC and 8 EC). 10 cc whole blood were devoted to PBMCs extraction (7 cc) and ScreenCell™ filters (3 cc) CTCs evaluation. Ficoll-isolated patient's PBMCs were seeded over CLG and allowed to infiltrate for 16 h; gels were digested and fixed with 2 % FA-HAse, cells stained and DAPI+/CD45-/pan-CK + cells enumerated as CTCs. Results: Human cancer cells infiltrate CLG more efficiently than EG (CLG/EG ratio 1.25 for HT29/1.58 for A498/1.71 for H460 and 2.83 for OVCAR8). CLG-recovered HT29 cells display hybrid-mesenchymal features [low E-cadherin (40 %) and high vimentin (235 %) as compared to HT29], CXCR4 two-fold higher than HT29, efficiently migrate toward CXCL12 (two-fold higher than HT29) and developed higher number of colonies (171 ± 21 for HT29-CLG vs 131 ± 8 colonies for HT29)/larger spheres (spheroid area: 26561 ± 6142 µm2 for HT29-CLG vs 20297 ± 7238 for HT29). In TRAP4MET clinical trial, CLG-CTCs were isolated in 8/8 patients with OC, 6/8 with LC, 6/8 with CRC, 8/8 with EC, 8/8 with RCC cancer and 5/8 with GBM. Interestingly, in OC, LC and GBM, CLG isolated higher number of CTCs as compared to the conventional ScreenCell™ (CLG/SC ratio = 1.88 for OC, 2.47 for LC and 11.89 for GBM). Bland and Altman blot analysis and Passing and Bablok regression analysis showed concordance between the methodological approaches but indicate that SC and CLG are not superimposable suggesting that the two systems select cells with different features. Conclusion: CLG might represent a new and easy tool to isolate invasive CTCs in multiple cancers such as OC, LC and GBM at today orphan of reliable methods to consistently detect CTCs.
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Introduction: DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Methods: Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. Results: We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. Discussion: We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.
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We previously demonstrated that the epidermal growth factor receptor (EGFR) modulates in mesenchymal stem cells (MSCs) the expression of a number of genes coding for secreted proteins that promote breast cancer progression. However, the role of the EGFR in modulating in MSCs the expression of miRNAs potentially involved in the progression of breast cancer remains largely unexplored. Following small RNA-sequencing, we identified 36 miRNAs differentially expressed between MSCs untreated or treated with the EGFR ligand transforming growth factor α (TGFα), with a fold change (FC) < 0.56 or FC ≥ 1.90 (CI, 95%). KEGG analysis revealed a significant enrichment in signaling pathways involved in cancer development and progression. EGFR activation in MSCs downregulated the expression of different miRNAs, including miR-23c. EGFR signaling also reduced the secretion of miR-23c in conditioned medium from MSCs. Functional assays demonstrated that miR-23c acts as tumor suppressor in basal/claudin-low MDA-MB-231 and MDA-MB-468 cells, through the repression of IL-6R. MiR-23c downregulation promoted cell proliferation, migration and invasion of these breast cancer cell lines. Collectively, our data suggested that the EGFR signaling regulates in MSCs the expression of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cell cross-talk.
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Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disorder and a complication of SARS-CoV-2 infection. In patients with severe SARS-CoV-2 infection, the transition to ARDS is principally due to the occurrence of a cytokine storm and an exacerbated inflammatory response. The effectiveness of ultra-micronized palmitoylethanolamide (PEA-um) during the earliest stage of COVID-19 has already been suggested. In this study, we evaluated its protective effects as well as the effectiveness of its congener, 2-pentadecyl-2-oxazoline (PEA-OXA), using in vitro models of acute lung injury. In detail, human lung epithelial cells (A549) activated by polyinosinic-polycytidylic acid (poly-(I:C)) or Transforming Growth Factor-beta (TGF-ß) were treated with PEA-OXA or PEA. The release of IL-6 and the appearance of Epithelial-Mesenchymal Transition (EMT) were measured by ELISA and immunofluorescence assays, respectively. A possible mechanism of action for PEA-OXA and PEA was also investigated. Our results showed that both PEA-OXA and PEA were able to counteract poly-(I:C)-induced IL-6 release, as well as to revert TGF-ß-induced EMT. In addition, PEA was able to produce an "entourage" effect on the levels of the two endocannabinoids AEA and 2-AG, while PEA-OXA only increased PEA endogenous levels, in poly-(I:C)-stimulated A549 cells. These results evidence for the first time the superiority of PEA-OXA over PEA in exerting protective effects and point to PEA-OXA as a new promising candidate in the management of acute lung injury.
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Lesão Pulmonar Aguda , COVID-19 , Humanos , Interleucina-6 , SARS-CoV-2 , Fator de Crescimento Transformador beta , Lesão Pulmonar Aguda/tratamento farmacológicoRESUMO
BACKGROUND AND PURPOSE: Airway remodelling is a critical feature of chronic lung diseases. Epithelial-mesenchymal transition (EMT) represents an important source of myofibroblasts, contributing to airway remodelling. Here, we investigated the sphingosine-1-phosphate (S1P) role in EMT and its involvement in asthma-related airway dysfunction. EXPERIMENTAL APPROACH: A549 cells were used to assess the S1P effect on EMT and its interaction with TGF-ß signalling. To assess the S1P role in vivo and its impact on lung function, two experimental models of asthma were used by exposing BALB/c mice to subcutaneous administration of either S1P or ovalbumin (OVA). KEY RESULTS: Following incubation with TGF-ß or S1P, A549 acquire a fibroblast-like morphology associated with an increase of mesenchymal markers and down-regulation of the epithelial. These effects are reversed by treatment with the TGF-ß receptor antagonist LY2109761. Systemic administration of S1P to BALB/c mice induces asthma-like disease characterized by mucous cell metaplasia and increased levels of TGF-ß, IL-33 and FGF-2 within the lung. The bronchi harvested from S1P-treated mice display bronchial hyperresponsiveness associated with overexpression of the mesenchymal and fibrosis markers and reduction of the epithelial.The S1P-induced switch from the epithelial toward the mesenchymal pattern correlates to a significant increase of lung resistance and fibroblast activation. TGF-ß blockade, in S1P-treated mice, abrogates these effects. Finally, inhibition of sphingosine kinases by SK1-II in OVA-sensitized mice, abrogates EMT, pulmonary TGF-ß up-regulation, fibroblasts recruitment and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS: Targeting S1P/TGF-ß axis may hold promise as a feasible therapeutic target to control airway dysfunction in asthma.
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Asma , Transição Epitelial-Mesenquimal , Esfingosina , Fator de Crescimento Transformador beta , Remodelação das Vias Aéreas , Animais , Asma/metabolismo , Asma/patologia , Células Epiteliais , Lisofosfolipídeos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment. METHODS: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells. RESULTS: We showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostat-inducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients. CONCLUSIONS: Overall, we suggest a novel therapeutic strategy based on domatinostat to improve efficacy and to overcome resistance of commonly used chemotherapeutics in PDAC that warrant further clinical evaluation.
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Benzamidas , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Benzamidas/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMO
Here, we tested whether co-targeting of glucose metabolism and oncogene drivers may enhance tumor response to tyrosine kinase inhibitors (TKIs) in NSCLC. To this end, pyruvate dehydrogenase kinase 1 (PDK1) was stably downregulated in oncogene-driven NSCLC cell lines exposed or not to TKIs. H1993 and H1975 cells were stably transfected with scrambled (shCTRL) or PDK1-targeted (shPDK1) shRNA and then treated with MET inhibitor crizotinib (1 µM), double mutant EGFRL858R/T790M inhibitor WZ4002 (1 µM) or vehicle for 48 h. The effects of PDK1 knockdown on glucose metabolism and apoptosis were evaluated in untreated and TKI-treated cells. PDK1 knockdown alone did not cause significant changes in glycolytic cascade, ATP production and glucose consumption, but it enhanced maximal respiration in shPDK1 cells when compared to controls. When combined with TKI treatment, PDK1 downregulation caused a strong enhancement of OXPHOS and a marked reduction in key glycolytic enzymes. Furthermore, increased levels of apoptotic markers were found in shPDK1 cells as compared to shCTRL cells after treatment with TKIs. Co-immunoprecipitation studies showed that PDK1 interacts with PKM2, Bcl-2 and Bcl-xL, forming macromolecular complexes at the ER-mitochondria interface. Our findings showed that downregulation of PDK1 is able to potentiate the effects of TKIs through the disruption of macromolecular complexes involving PKM2, Bcl-2 and Bcl-xL.
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Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.
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Malignant mesothelioma (MM) is an aggressive asbestos-related cancer, against which no curative modalities exist. Oncolytic virotherapy is a promising therapeutic approach, for which MM is an ideal candidate; indeed, the pleural location provides direct access for the intra-tumoral injection of oncolytic viruses (OVs). Some non-human OVs offer advantages over human OVs, including the non-pathogenicity in humans and the absence of pre-existing immunity. We previously showed that caprine herpesvirus 1 (CpHV-1), a non-pathogenic virus for humans, can kill different human cancer cell lines. Here, we assessed CpHV-1 effects on MM (NCI-H28, MSTO, NCI-H2052) and non-tumor mesothelial (MET-5A) cells. We found that CpHV-1 reduced cell viability and clonogenic potential in all MM cell lines without affecting non-tumor cells, in which, indeed, we did not detect intracellular viral DNA after treatment. In particular, CpHV-1 induced MM cell apoptosis and accumulation in G0/G1 or S cell cycle phases. Moreover, CpHV-1 strongly synergized with cisplatin, the drug currently used in MM chemotherapy, and this agent combination did not affect normal mesothelial cells. Although further studies are required to elucidate the mechanisms underlying the selective CpHV-1 action on MM cells, our data suggest that the CpHV-1-cisplatin combination could be a feasible strategy against MM.
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Antineoplásicos/farmacologia , Apoptose , Cisplatino/farmacologia , Mesotelioma Maligno/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Varicellovirus/fisiologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Humanos , Mesotelioma Maligno/tratamento farmacológico , Mesotelioma Maligno/fisiopatologia , Mesotelioma Maligno/virologia , Vírus Oncolíticos/genética , Varicellovirus/genéticaRESUMO
The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.
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Micropartículas Derivadas de Células/metabolismo , Lisossomos/metabolismo , Neovascularização Patológica/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Sarcoma/irrigação sanguínea , Sarcoma/patologia , Animais , Cálcio/metabolismo , Movimento Celular , Citosol/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Retina/patologia , Sarcoma/sangue , Transdução de Sinais , ViscosidadeRESUMO
Lung cancer (LC) is the main cause of death for cancer worldwide and non-small cell lung cancer (NSCLC) represents the most common histology. The discovery of genomic alterations in driver genes that offer the possibility of therapeutic intervention has completely changed the approach to the diagnosis and therapy of advanced NSCLC patients, and tumor molecular profiling has become mandatory for the choice of the most appropriate therapeutic strategy. However, in approximately 30% of NSCLC patients tumor tissue is inadequate for biomarker analysis. The development of highly sensitive next generation sequencing (NGS) technologies for the analysis of circulating cell-free DNA (cfDNA) is emerging as a valuable alternative to assess tumor molecular landscape in case of tissue unavailability. Additionally, cfDNA NGS testing can better recapitulate NSCLC heterogeneity as compared with tissue testing. In this review we describe the main advantages and limits of using NGS-based cfDNA analysis to guide the therapeutic decision-making process in advanced NSCLC patients, to monitor the response to therapy and to identify mechanisms of resistance early. Therefore, we provide evidence that the implementation of cfDNA NGS testing in clinical research and in the clinical practice can significantly improve precision medicine approaches in patients with advanced NSCLC.
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This study explores the possibility to use the extremophilic microalga Galdieria sulphuraria (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. Galdieria was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. Galdieria extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that G. sulphuraria from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Rodófitas/metabolismo , Células A549 , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Glutationa/análise , Processos Heterotróficos , Humanos , Ficocianina/análise , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Rodófitas/química , Rodófitas/citologiaRESUMO
The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-ß1 quantification. We found that LC212-CM contains high levels of TGF-ß1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-ß1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-ß1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-ß1. In conclusion, hsa-miR21 and TGF-ß1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células A549 , Forma Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , FenótipoRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p = 0.05) and DRB1*01 (p = 0.05) with the linked DQB1*0501 (p = 0.01) alleles, has been observed. Notably, a fourfold increase of the haplotype B*1402, Cw*0802 (p < 0.0005) and a 15-fold increase of the Mediterranean haplotype A*33, B*1402, Cw*0802, DRB1*0102, DQB1*0501 (p < 0.005) was also revealed. This association may provide new insights into the autoimmune pathogenesis of PNH.