RESUMO
Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Metabolismo Energético , Neoplasias Pulmonares , Mitocôndrias , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Ácidos Graxos/metabolismo , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Fenótipo , Tomografia por Emissão de PósitronsRESUMO
The neuroendocrine transdifferentiation has been found in many cancer cell types, such as prostate, lung and gastrointestinal cells and is accompanied by a lower patient life expectancy. The transdifferentiation process has been induced in vitro by the exposure to different stimuli in human lung adenocarcinoma. The aim of this work was to identify the morphological characteristics of the neuroendocrine phenotype in a human lung cancer cell line, induced by two cAMP elevating agents (IBMX and FSK). Our results showed two phenotypes, one produced by IBMX with higher volume, cell size and increased number of secondary projections, and the other produced by FSK with higher area, roughness of the membrane, cell neurite percentage, number of outgrowths per cell and increased number of primary projections. In conclusion, we describe some morphological and ultrastructural characteristics of the neuroendocrine phenotype in A549 human lung cancer cell line promoted by IBMX and FSK to contribute to the understanding of the autocrine or paracrine signaling within the tumor microenvironment.
Assuntos
Adenocarcinoma de Pulmão , Transdiferenciação Celular , Neoplasias Pulmonares , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/ultraestrutura , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestrutura , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/ultraestruturaRESUMO
While embryonic stem cells and cancer cells are known to have many similarities in signalling pathways, healthy somatic cells are known to be different in many ways. Characterization of embryonic stem cell is crucial for cancer development and cancer recurrence due to the shared signalling pathways and life course with cancer initiator and cancer stem cells. Since embryonic stem cells are the sources of the somatic and cancer cells, it is necessary to reveal the relevance between them. The past decade has seen the importance of interdisciplinary studies and it is obvious that the reflection of the physical/chemical phenomena occurring on the cell biology has attracted much more attention. For this reason, the aim of this study is to elementally and topologically characterize the mouse embryonic stem cells, mouse lung squamous cancer cells, and mouse skin fibroblast cells by using Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM) supported with Electron Dispersive Spectroscopy (EDS) techniques in a complementary way. Our AFM findings revealed that roughness data of the mouse embryonic stem cells and cancer cells were similar and somatic cells were found to be statistically different from these two cell types. However, based on both XPS and SEM-EDS results, surface elemental ratios vary in mouse embryonic stem cells, cancer cells and somatic cells. Our results showed that these complementary spectroscopic and microscopic techniques used in this work are very effective in cancer and stem cell characterization and have the potential to gather more detailed information on relevant biological samples.
Assuntos
Fibroblastos , Neoplasias Pulmonares , Células-Tronco Embrionárias Murinas , Neoplasias de Células Escamosas , Pele , Animais , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/ultraestrutura , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/ultraestrutura , Pele/metabolismo , Pele/ultraestruturaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Feiyanning (FYN), the Chinese herbal medicine (CHM), has been used to manage non-small cell lung cancer (NSCLC) for the past 23 years. Chemotherapeutic drugs can induce autophagy in cancer cells to protect themselves from death. However, FYN can inhibit the protective autophagy in cancer cells. We investigated the biological mechanisms on the synergistic effects of FYN combined with chemotherapy in lung cancer cells. MATERIALS AND METHODS: We analyzed the effective chemical components for the quality control of FYN using the UPLC-Q-TOF-MS.The cell proliferation ability was detected by the cell counting kit-8 (CCK-8) and colony formation. The cell apoptosis was determined with Flow cytometry. Expression of important differential proteins were detected by western blot. Autophagy structure was observed by TEM (Tansmission electron microscopy). Tandem mCherry-EGFP-LC3B immunofluorescence was used to measure autophagic flux. RESULTS: Both FYN and cisplatin significantly induced apoptosis and inhibited cell proliferation in A549 cells. FYN reduced cell viability and increased apoptotic cell populations less effectively than cisplatin. FYN cooperated with cisplatin suppressed the cell viability, colony formation, as well as increased the cell apoptosis rate, and the expression of cleaved caspase-3 and PARP. FYN inhibited autophagy in A549 cells, which characterized by the decrease of autophagosome formation, lysosomal fusion, LC3B-II accumulation and SQSTM1 degradation, down-regulation of ATG5 and ATG7. Protective autophagy in A549 cells was induced by cisplatin. Suppression of the autophagic response using chloroquine (CQ) which is autophagy inhibitor improved the ability of cisplatin to kill cancer cells, as did FYN combined with cisplatin. CONCLUSION: In summary, we revealed that the synergistic mechanism of FYN and cisplatin is that FYN inhibited the protective autophagy induced by cisplatin in A549 cells.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/química , Humanos , Neoplasias Pulmonares/ultraestruturaRESUMO
PURPOSE: MicroRNAs act as crucial regulators of a diverse range of biological processes, including chemoresistance. Our study aimed to investigate the effect of miR-324-3p on lung adenocarcinoma cell line A549 resistant to cis-diamminedichloroplatinum II (DDP, aka cisplatin). METHODS: The miR-324-3p expression levels in cisplatin-sensitive A549ï¼A549ï¼ and cisplatin-resistant A549 (A549/DDP) cells were determined by qRT-PCR assay. Cell proliferation was determined with the commercial kit CCK-8 and colony formation assay, whereas cell death was analyzed using flow cytometry. The target gene of miR-324-3p was identified and validated with the luciferase reporter and western blot assays. The role of miR-324-3p in modulating cisplatin resistance was evaluated in vitro. RESULTS: The expression of miR-324-3p was found to be significantly downregulated in the A549/DDP cells. Conversely, miR-324-3p overexpression reversed cisplatin resistance in the cells. With regard to the possible mechanism underlying this phenomenon, we identified the glutathione peroxidase 4 (GPX4) gene as the direct target of miR-324-3p, where overexpression of the gene reversed the miR-324-3p effect of sensitizing the A549/DDP cells to cisplatin. Furthermore, the GPX4 inhibitor RSL3 could mimic the effect of miR-324-3p upregulation in increasing the sensitivity of the cisplatin-resistant cells to the drug. Significantly, miR-324-3p enhanced cisplatin-induced ferroptosis in the A549/DDP cells. CONCLUSION: Our findings revealed the role of the miR-324-3p-GPX4 signaling axis in A549/DDP cells and how the targeting of this axis could be a potential strategy for reversing cisplatin resistance in human non small cell lung cancer (NSCLC).
Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ferroptose/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Células A549 , Adenocarcinoma de Pulmão/ultraestrutura , Sequência de Bases , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , MicroRNAs/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through "T"-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Interpretação de Imagem Assistida por Computador , Imageamento Tridimensional , Membranas Intracelulares/ultraestrutura , Neoplasias Pulmonares/ultraestrutura , Organelas/ultraestrutura , Alvéolos Pulmonares/ultraestrutura , Células A549 , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Organelas/metabolismo , Alvéolos Pulmonares/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
PURPOSE: Paclitaxel is an anticancer drug for the treatment of non-small cell lung cancer (NSCLC). However, drug-resistance remains a major problem. Honokiol is a natural component which has been found to exhibit anti-tumor activity. Paclitaxel and honokiol have been reported to be able to induce paraptosis. The aim of this study was to investigate whether honokiol can reverse paclitaxel resistance by inducing paraptosis in NSCLC cells. METHODS: NSCLC cell lines H1650 (paclitaxel-sensitive), H1299 and H1650/PTX (intrinsic and acquired paclitaxel-resistant, respectively) were used to assess the cytotoxic effects of paclitaxel and honokiol. Light and transmission electron microscopy were performed to detect cytoplasmic vacuolation. In vitro cell viability and clonogenic survival assays, as well as in vivo xenograft assays were conducted to test synergistic killing effects of paclitaxel and honokiol on NSCLC cells. Western blotting, flow cytometry and immunofluorescence were performed to evaluate paraptosis-regulating mechanisms. RESULTS: We found that combination treatment with paclitaxel and honokiol synergistically killed H1650, H1299 and H1650/PTX cells by inducing paraptosis, which is characterized by cytoplasmic vacuolation. Moreover, paclitaxel/honokiol treatment resulted in a significant growth delay in H1299 xenograft tumors that showed extensive cytoplasmic vacuolation. Mechanistically, proteasomal inhibition-mediated endoplasmic reticulum (ER) stress and unfolded protein responses leading to ER dilation, and the disruption of intracellular Ca2+ homeostasis and mitochondrial Ca2+ overload resulting in mitochondrial disfunction, were found to be involved in paclitaxel/honokiol-induced paraptosis. Cellular protein light chain 3 (LC3) may play an important role in paclitaxel/honokiol induced cytoplasmic vacuolation and NSCLC cell death. CONCLUSIONS: Combination of honokiol and paclitaxel may represent a novel strategy for the treatment of paclitaxel-resistant NSCLC.
Assuntos
Apoptose , Compostos de Bifenilo/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Lignanas/farmacologia , Neoplasias Pulmonares/patologia , Paclitaxel/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Linhagem Celular Tumoral , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
The aim of the present study was to unveil the chemopreventive potentials of aqueous Tinospora cordifolia stem extract and its active component viz. Arabinogalactan against Benzo(a)pyrene-induced pulmonary carcinogenesis. Animals were divided into six groups: (I) Control, (II) aqueous Tinospora cordifolia (200 mg/kg b.wt, p.o.), (III) arabinogalactan (7.5 mg/kg b.wt, p.o.), (IV) benzo(a)pyrene (50 mg/kg b.wt, i.p.) at second and fourth week of study, (V) benzo(a)pyrene + aqueous Tinospora cordifolia, and (VI) benzo(a)pyrene + arabinogalactan. The benzo(a)pyrene treatment resulted in severe alterations in the cellular arrangement and morphology of the alveolar tissue in benzo(a)pyrene group. However, benzo(a)pyrene + aqueous Tinospora cordifolia and benzo(a)pyrene + arabinogalactan groups revealed classical features of apoptosis including chromatin condensation and formation of apoptotic bodies. Furthermore, Fourier transform Infrared spectroscopy analysis showed disturbed phospholipid saturation and protein secondary structures in benzo(a)pyrene treated animals. Depletion in relative glycogen and enhancement in total nucleic acid content was observed in benzo(a)pyrene treated animals, and the same was found to be restored upon arabinogalactan and aqueous Tinospora cordifolia supplementation. Benzo(a)pyrene insult also upregulated the phase I carcinogen metabolizing enzymes and differentially modulated the phase II metabolizing enzymes during pulmonary carcinogenesis. Also, depleted (reduced glutathione) and increased lipid peroxidation levels were observed in benzo(a)pyrene treated animals, which was found to be normalized upon aqueous Tinospora cordifolia and arabinogalactan administration. Clastogenic damage inflicted by benzo(a)pyrene was also reversed in benzo(a)pyrene + aqueous Tinospora cordifolia and benzo(a)pyrene + arabinogalactan group. Thus, the present study infers that aqueous Tinospora cordifolia and arabinogalactan showed promising anticancer activity against lung tumorigenesis in terms of ultrastructural, biochemical, and biomolecular aspects.
Assuntos
Benzo(a)pireno/toxicidade , Carcinogênese/efeitos dos fármacos , Galactanos/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Tinospora , Carcinogênese/induzido quimicamente , Quimioprevenção , Humanos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestruturaRESUMO
Autophagy inhibition has been demonstrated to increase the efficacy of conventional chemotherapy. In this study, we identified hederagenin, a triterpenoid derived from Hedera helix, as a potent inhibitor of autophagy and then hypothesized that hederagenin might synergize with chemotherapeutic drugs (e.g., cisplatin and paclitaxel) to kill lung cancer cells. Firstly, we observed that hederagenin induced the increased autophagosomes in lung cancer cells concomitantly with the upregulation of LC3-II and p62, which indicated the impairment of autophagic flux. The colocalization assay indicated hederagenin could not block the fusion of lysosomes and autophagosomes, whereas the lysosomal acidification might be inhibited by hederagenin as revealed by the reduced staining of acidity-sensitive reagents (i.e., Lysotracker and acridine orange). The aberrant acidic environment then impaired the function of lysosome, which was evidenced by the decrease of mature cathepsin B and cathepsin D. Lastly, hederagenin, in agree with our hypothesis, promoted pro-apoptotic effect of cisplatin and paclitaxel with the accumulation of reactive oxygen species (ROS); while the synergistic effect could be abolished by the ROS scavenger, N-acetyl-L-cysteine. These data summarily demonstrated hederagenin-induced accumulation of ROS by blocking autophagic flux potentiated the cytotoxicity of cisplatin and paclitaxel in lung cancer cells.
Assuntos
Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Pulmonares/patologia , Ácido Oleanólico/análogos & derivados , Paclitaxel/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, we identified a circular form of ASPH RNA (circASPH), expression of which was upregulated in lung adenocarcinoma and the human lung adenocarcinoma cell lines. We also found a positive correlation between circASPH level and the T and N stages of lung adenocarcinoma patients. Patients with higher levels of circASPH had a shorter overall survival. Moreover, we demonstrated that circASPH was directly regulated by HMGA2 and Twist1. The direct positive regulation of circASPH by Twist1 was dependent on the presence of HMGA2. Functional assays indicated that circASPH promoted the proliferation, migration, and invasion of lung adenocarcinoma cell lines in vitro. The promoting effect of tumor growth by circASPH was also observed in vivo. Mechanistically, circASPH was identified to act as a molecular sponge for miR-370 and abrogate miR-370-mediated inhibition of HMGA2. Finally, we demonstrated that the oncogenic function of circASPH was HMGA2-dependent. These findings reveal the oncogenic functions of the HMGA2-circASPH-HMGA2 axis and may be useful in developing circRNA-based therapeutic strategies for lung adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Regulação Neoplásica da Expressão Gênica , Proteína HMGA2/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , RNA Circular/genética , Adenocarcinoma de Pulmão/ultraestrutura , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/ultraestrutura , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , RNA Circular/metabolismo , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Y-box binding protein 1 (YBX1) is involved in the development of multiple types of tumors. However, the relationship between YBX1 and autophagy in non-small cell lung cancer (NSCLC) remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and markers of autophagy (LC3I/II) in NSCLC and examined their roles in regulating sensitivity to cisplatin in NSCLC. The retrospective analysis of patients with NSCLC indicated that YBX1 was positively correlated with autophagy. Increased levels of YBX1 or autophagy also observed in NSCLC cells compared with those in 16HBE cells. Compared to the controls, the knockdown of YBX1 expression suppressed autophagy, increased drug sensitivity and promoted apoptosis in response to cisplatin in NSCLC cells by targeting the p110ß promoter and inhibiting p110ß/Vps34/beclin1 signaling pathways. We also demonstrated in an in vivo study that the overexpressed YBX1 effectively increased NSCLC growth and progression and decreased the sensitivity to cisplatin by inducing autophagy in a xenograft tumor model, and these effects were concomitant with the increasing of p110ß and beclin1 expression. Collectively, these results show that YBX1 plays an essential role in autophagy in NSCLC.
Assuntos
Autofagia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/urina , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteína 1 de Ligação a Y-Box/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-IdadeRESUMO
Anoikis (detachment-induced cell death) is a specific type of programmed cell death which occurs in response to the loss of the correct extracellular matrix connections. Anoikis resistance is an important mechanism in cancer invasiveness and metastatic behavior. Autophagy, on the other hand, involves the degradation of damaged organelles and the recycling of misfolded proteins and intracellular components. However, the intersection of these two cellular responses in lung cancer cells has not been extensively studied. Here, we identified that upon matrix deprivation, the lymphocyte lineage-specific Ets transcription factor SPIB was activated and directly enhanced SNAP47 transcription in certain lung cancer cells. Loss of attachment-induced autophagy significantly increased anoikis resistance by SPIB activation. Consistent with this function, SPIB depletion by short hairpin RNA abrogated SNAP47 transcriptional activation upon matrix deprivation. Therefore, these data delineate an important role of SPIB in autophagy-mediated anoikis resistance in lung cancer cells. Accordingly, these findings suggest that manipulating SPIB-regulated pathways in vivo and evaluating the impact of anoikis resistance warrant further investigation. DATABASE: RNA sequencing and ChIP sequencing data are available in Gene Expression Omnibus database under the accession numbers GSE106592 and GSE125561, respectively.
Assuntos
Anoikis/genética , Autofagia/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Lisossomos/metabolismo , Fatores de Transcrição/genética , Células A549 , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestrutura , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Under the National Comprehensive Cancer Network (NCCN) guidelines for non-small-cell lung carcinoma (NSCLC), anaplastic lymphoma kinase (ALK) gene rearrangement is required to be assessed. However, data showing the prevalence of the ALK rearrangement is still deficient and is not yet available in Indonesia. This study used direct smear preparation from transthoracic needle specimens that are minimally invasive. The main objective of the study is to identify the prevalence of the ALK fusion rearrangement gene in cytological specimens. MATERIALS AND METHODS: A total of 35 direct smear preparations diagnosed as lung adenocarcinoma and EGFR mutation negative were involved in this study. The samples were taken between 2017 and 2019. These samples were examined for EML4-ALK fusion rearrangement gene using qRT-PCR. The EML4-ALK rearrangement status was determined by qRT-PCR with high-resolution melting (HRM) analysis. RESULTS: A total of 28 (80%) samples were from males, and 7 samples were from females. Seven (20% 95% CI: 8.4%-36.9%) samples were EML4-ALK rearrangement positive. The average age of the patients was 63.5 years old. The most common sites of metastasis in this study were pleural cavity, bone, liver, and CNS. CONCLUSIONS: qRT-PCR successfully identified EML4-ALK fusion rearrangement in direct smear preparations of lung adenocarcinoma. Direct smear samples can be used for EML4-ALK rearrangement detection using qRT-PCR. The EML4-ALK rearrangement gene has high prevalence in selected lung adenocarcinoma and EGFR mutation-negative populations. ALK inhibitors in lung cancer can be openly considered for use in Indonesian patients to improve the outcome of this subset of patients.
Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Adenocarcinoma de Pulmão/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Indonésia , Pulmão/ultraestrutura , Neoplasias Pulmonares/ultraestrutura , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/ultraestrutura , PrevalênciaRESUMO
Rationale: Lung cancer is the leading cause of cancer death worldwide, and treatment options are limited to mainly cytotoxic agents. Here we reveal a novel role of miR-150 in non-small cell lung cancer (NSCLC) development and seek potential new therapeutic targets. Methods: The miR-150-mediated autophagy dysfunction in NSCLC cells were examined using molecular methods in vitro and in vivo. The upstream regulatory element and downstream target of miR-150 were identified in vitro and validated in vivo. Potential therapeutic methods (anti-c-myc or anti-miR-150) were tested in vitro and in vivo. Clinical relevance of the c-myc/miR-150/EPG5 axis in NSCLC was validated in human clinical samples and large genomics database. Results: miR-150 blocked the fusion of autophagosomes and lysosomes through directly repressing EPG5. The miR-150-mediated autophagy defect induced ER stress and increased cellular ROS levels and DNA damage response, and promoted NSCLC cell proliferation and tumor growth. Knockdown of EPG5 promoted NSCLC cell proliferation, and attenuated the effects of miR-150. c-myc gene was identified as a miR-150 transcriptional factor which increased miR-150 accumulation, therefore pharmacologically or genetically inhibiting c-myc/miR-150 expression significantly inhibited NSCLC cell growth in vitro and in vivo. Both c-myc and miR-150 were significantly over-expressed in NSCLC, while EPG5 was down-regulated in NSCLC. Expression levels of these molecules were well correlated, and also well correlated with patient survival. Conclusions: Our findings suggest that c-myc/miR-150/EPG5 mediated dysfunction of autophagy contributes to NSCLC development, which may provide a potential new diagnostic and therapeutic target in NSCLC.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Progressão da Doença , Estresse do Retículo Endoplasmático/genética , Feminino , Inativação Gênica , Humanos , Neoplasias Pulmonares/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Fusão de Membrana , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Análise de SobrevidaRESUMO
Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4',6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Colorretais/patologia , Molécula de Adesão da Célula Epitelial/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Contagem de Células , Vesículas Extracelulares/patologia , Feminino , Humanos , Leucócitos/metabolismo , Neoplasias Pulmonares/ultraestruturaRESUMO
Magnetotactic bacteria are aquatic microorganisms that internally biomineralize chains of magnetic nanoparticles (called magnetosomes) and use them as a compass. Here it is shown that magnetotactic bacteria of the strain Magnetospirillum gryphiswaldense present high potential as magnetic hyperthermia agents for cancer treatment. Their heating efficiency or specific absorption rate is determined using both calorimetric and AC magnetometry methods at different magnetic field amplitudes and frequencies. In addition, the effect of the alignment of the bacteria in the direction of the field during the hyperthermia experiments is also investigated. The experimental results demonstrate that the biological structure of the magnetosome chain of magnetotactic bacteria is perfect to enhance the hyperthermia efficiency. Furthermore, fluorescence and electron microscopy images show that these bacteria can be internalized by human lung carcinoma cells A549, and cytotoxicity studies reveal that they do not affect the viability or growth of the cancer cells. A preliminary in vitro hyperthermia study, working on clinical conditions, reveals that cancer cell proliferation is strongly affected by the hyperthermia treatment, making these bacteria promising candidates for biomedical applications.
Assuntos
Hipertermia Induzida , Campos Magnéticos , Magnetospirillum/fisiologia , Células A549 , Sobrevivência Celular , Fluorescência , Humanos , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/ultraestrutura , Magnetossomos/química , Magnetossomos/ultraestrutura , Magnetospirillum/ultraestrutura , Temperatura , Fatores de TempoRESUMO
BACKGROUND: Polymeric delivery systems have been elucidated over the last few years as an approach of achieving high therapeutic effect to the local site of malignant disease patients who have cancer. Polypyrrole (Ppy) is a potential organic conducting polymer which has long been recognized as a versatile material due to its excellent stability, conductive properties, and great absorbance in the range of near-infrared (NIR). It is tremendously versatile for use in various biomedical fields such as cancer therapy. NIR irradiation-activated treatment platform technologies are now being considered to be novel and exciting options in potential nanomedicine. However, the realistic photothermal use of Ppy-applied nanomaterials is yet in its early phase, and there are a few disadvantages of Ppy, such as its water insolubility. In the clinic, the common approach for treatment of lung cancer is the delivery of therapeutic active substances through intratumoral administration. Nevertheless, the tumor uptake, regional retention, mechanism of treatment, and tissue organ penetration regarding the developed strategy of this nanomaterial with photothermal hyperthermia are important issues for exerting effective cancer therapy. MATERIALS AND METHODS: In this study, we developed a cationic Ppy-polyethylenimine nanocomplex (NC) with photothermal hyperthermia to study its physicochemical characteristics, including size distribution, zeta potential, and transmission electron microscopy, scanning electron microscopy, and Fourier transform infrared morphology. We also examined the cellular uptake effect on lung cancer cells, the photothermal properties, intracellularly generated reactive oxygen species (ROS), and cytotoxicity. RESULTS: The results suggested that this nanocarrier system was able to effectively attach onto lung cancer cells for subsequent endocytosis. The NCs taken up were able to absorb NIR and then converted the NIR light into local hyperthermia with its intracellular photothermal performance to provide local hyperthermic treatment. This regionally generated hyperthermia also induced ROS formation and improved the killing of lung cancer cells as a promising local photothermal therapy. CONCLUSION: This development of a nanocarrier would bring a novel therapeutic strategy for lung cancer in the future.
Assuntos
Hipertermia Induzida , Neoplasias Pulmonares/terapia , Nanoestruturas/química , Fototerapia , Polietilenoimina/química , Polímeros/química , Pirróis/química , Espécies Reativas de Oxigênio/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Fluorescência , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Nanoestruturas/ultraestrutura , Propriedades de SuperfícieRESUMO
Hirsutine extracted from Uncaria rhynchophylla has been shown to exhibit anti-cancer activity. However, the molecular mechanism by which hirsutine exhibits anti-lung cancer activity remains unclear. In the present study, we showed that hirsutine induces apoptosis in human lung cancer cells via loss of mitochondrial membrane potential (∆ψm), adenosine triphosphate (ATP) depletion, ROS production, as well as cytochrome c release. Dephosphorylation of GSK3ß is involved in hirsutine-mediated mitochondrial permeability transition pore (mPTP) opening through ANT1/CypD interaction. Mechanistic study revealed that interruption of ROCK1/PTEN/PI3K/Akt signaling pathway plays a critical role in hirsutine-mediated GSK3ß dephosphorylation and mitochondrial apoptosis. Our in vivo study also showed that hirsutine effectively inhibits tumor growth in a A549 xenograft mouse model through ROCK1/PTEN/PI3K/Akt signaling-mediated GSK3ß dephosphorylation and apoptosis. Collectively, these findings suggest a hierarchical model in which induction of apoptosis by hirsutine stems primarily from activation of ROCK1 and PTEN, inactivation of PI3K/Akt, leading in turn to GSK3ß dephosphorylation and mPTP opening, and culminating in caspase-3 activation and apoptosis. These findings could provide a novel mechanistic basis for the application of hirsutine in the treatment of human lung cancer.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transdução de Sinais , Células A549 , Translocador 1 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Alcaloides/química , Animais , Proliferação de Células/efeitos dos fármacos , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Citocromos c/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Poro de Transição de Permeabilidade Mitocondrial , Modelos Biológicos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: Leukocyte telomere length (LTL) is a potential biomarker of cancer prognosis; however, evidence for renal cell carcinoma (RCC) is inconsistent. METHODS: We investigated LTL and RCC-specific survival among 684 cases from the US kidney cancer study (USKC) and 241 cases from the prostate, lung, colorectal, and ovarian cancer screening trial (PLCO). Leukocyte telomere length was measured by quantitative polymerase chain reaction, and hazard ratios (HRs) and 95% confidence intervals (CIs) computed using multivariable Cox models. RESULTS: Short LTL was associated with poorer disease-specific survival in both USKC (lowest vs highest quartile: HR: 2.3, 95% CI: 1.2-4.4; P for trend=0.02) and PLCO (HR: 2.4, 95% CI: 1.0-5.4; P=0.04). Among USKC cases, the association was strongest for stage-I RCC (HR: 5.5, 95% CI: 1.6-19.0; P=0.006). CONCLUSIONS: Our findings suggest that shorter LTL is an independent marker of poor RCC prognosis, particularly for stage-I disease.