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1.
Nat Commun ; 12(1): 2038, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33795683

RESUMEN

Wild-type KRAS (KRASWT) amplification has been shown to be a secondary means of KRAS activation in cancer and associated with poor survival. Nevertheless, the precise role of KRASWT overexpression in lung cancer progression is largely unexplored. Here, we identify and characterize a KRAS-responsive lncRNA, KIMAT1 (ENSG00000228709) and show that it correlates with KRAS levels both in cell lines and in lung cancer specimens. Mechanistically, KIMAT1 is a MYC target and drives lung tumorigenesis by promoting the processing of oncogenic microRNAs (miRNAs) through DHX9 and NPM1 stabilization while halting the biogenesis of miRNAs with tumor suppressor function via MYC-dependent silencing of p21, a component of the Microprocessor Complex. KIMAT1 knockdown suppresses not only KRAS expression but also KRAS downstream signaling, thereby arresting lung cancer growth in vitro and in vivo. Taken together, this study uncovers a role for KIMAT1 in maintaining a positive feedback loop that sustains KRAS signaling during lung cancer progression and provides a proof of principle that interfering with KIMAT1 could be a strategy to hamper KRAS-induced tumorigenesis.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Largo no Codificante/genética , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
2.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802212

RESUMEN

Targetable alterations in cancer offer novel opportunities to the drug discovery process. However, pre-clinical testing often requires solubilization of these drugs in cosolvents like dimethyl sulfoxide (DMSO). Using a panel of cell lines commonly used for in vitro drug screening and pre-clinical testing, we explored the DMSO off-target effects on functional signaling networks, drug targets, and downstream substrates. Eight Non-Small Cell Lung Cancer (NSCLC) cell lines were incubated with three concentrations of DMSO (0.0008%, 0.002%, and 0.004% v/v) over time. Expression and activation levels of 187 proteins, of which 137 were kinases and downstream substrates, were captured using the Reverse Phase Protein Array (RPPA). The DMSO effect was heterogeneous across cell lines and varied based on concentration, exposure time, and cell line. Of the 187 proteins measured, all were statistically different in at least one comparison at the highest DMSO concentration, followed by 99.5% and 98.9% at lower concentrations. Only 46% of the proteins were found to be statistically different in more than 5 cell lines, indicating heterogeneous response across models. These cell line specific alterations modulate response to in vitro drug screening. Ultra-low DMSO concentrations have broad and heterogeneous effects on targetable signaling proteins. Off-target effects need to be carefully evaluated in pre-clinical drug screening and testing.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Dimetilsulfóxido/farmacología , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Transducción de Señal/efectos de los fármacos , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología
3.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-33802597

RESUMEN

Heat shock protein 90 (HSP90) plays an essential role in lung adenocarcinoma, acting as a key chaperone involved in the correct functioning of numerous highly relevant protein drivers of this disease. To this end, HSP90 inhibitors have emerged as promising therapeutic strategies, even though responses to them have been limited to date. Given the need to maximize treatment efficacy, the objective of this study was to use isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic techniques to identify proteins in human lung adenocarcinoma cell lines whose basal abundances were correlated with response to HSP90 inhibitors (geldanamycin and radicicol derivatives). From the protein profiles identified according to response, the relationship between lactate dehydrogenase B (LDHB) and DNA topoisomerase 1 (TOP1) with respect to sensitivity and resistance, respectively, to geldanamycin derivatives is noteworthy. Likewise, rhotekin (RTKN) and decaprenyl diphosphate synthase subunit 2 (PDSS2) were correlated with sensitivity and resistance to radicicol derivatives. We also identified a relationship between resistance to HSP90 inhibition and the p53 pathway by glucose deprivation. In contrast, arginine biosynthesis was correlated with sensitivity to HSP90 inhibitors. Further study of these outcomes could enable the development of strategies to improve the clinical efficacy of HSP90 inhibition in patients with lung adenocarcinoma.


Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Antineoplásicos/farmacología , Benzoquinonas/farmacología , Línea Celular Tumoral , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Chaperonas Moleculares/metabolismo , Proteómica/métodos
4.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807876

RESUMEN

In the scenario of systemic treatment for advanced non-small cell lung cancer (NSCLC) patients, one of the most relevant breakthroughs is represented by targeted therapies. Throughout the last years, inhibitors of the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-Ros oncogene 1 (ROS1), and V-raf murine sarcoma viral oncogene homolog B (BRAF) have been approved and are currently used in clinical practice. However, other promising molecular drivers are rapidly emerging as therapeutic targets. This review aims to cover the molecular alterations with a potential clinical impact in NSCLC, including amplifications or mutations of the mesenchymal-epithelial transition factor (MET), fusions of rearranged during transfection (RET), rearrangements of the neurotrophic tyrosine kinase (NTRK) genes, mutations of the Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), as well as amplifications or mutations of human epidermal growth factor receptor 2 (HER2). Additionally, we summarized the current status of targeted agents under investigation for such alterations. This revision of the current literature on emerging molecular targets is needed as the evolving knowledge on novel actionable oncogenic drivers and targeted agents is expected to increase the proportion of patients who will benefit from tailored therapeutic approaches.


Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas , Sistemas de Liberación de Medicamentos , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo
5.
Medicine (Baltimore) ; 100(14): e25415, 2021 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-33832139

RESUMEN

BACKGROUND: Circular RNAs (circRNAs) regulate multiple pathways during lung cancer pathogenesis. Apart from functional significance, many circRNAs have been shown to be associated with clinicopathological characteristics and predict lung cancer prognosis. Our aim is to summarize the expanding knowledge of clinical roles of circRNAs in lung cancer. METHODS: A thorough search of literature was conducted to identify articles about the correlation between circRNA expression and its prognostic and clinicopathological values. Biological mechanisms were summarized. RESULTS: This study included 35 original articles and 32 circRNAs with prognostic roles for lung cancer. Increased expression of 25 circRNAs and decreased expression of 7 circRNAs predicted poor prognosis. For non-small cell lung cancer, changes of circRNAs were correlated with tumor size, lymph node metastasis, distant metastasis, tumor node metastasis (TNM) stage, and differentiation, indicating the major function of circRNAs is to promote lung cancer invasion and migration. Particularly, meta-analysis of ciRS-7, hsa_circ_0020123, hsa_circ_0067934 showed increase of the 3 circRNAs was associated with positive lymph node metastasis. Increase of ciRS-7 and hsa_circ_0067934 was also related with advanced TNM stage. The biological effects depend on the general function of circRNA as microRNA sponge. CONCLUSIONS: CircRNAs have the potential to function as prognostic markers and are associated with lung cancer progression and metastasis.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/diagnóstico , ARN Circular/metabolismo , Progresión de la Enfermedad , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico
6.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33809929

RESUMEN

The occurrence of distant tumor metastases is a major barrier in non-small cell lung cancer (NSCLC) therapy, and seriously affects clinical treatment and patient prognosis. Recently, long non-coding RNAs (lncRNAs) have been demonstrated to be crucial regulators of metastasis in lung cancer. The aim of this study was to reveal the underlying mechanisms of a novel lncRNA LNC CRYBG3 in regulating NSCLC metastasis. Experimental results showed that LNC CRYBG3 was upregulated in NSCLC cells compared with normal tissue cells, and its level was involved in these cells' metastatic ability. Exogenously overexpressed LNC CRYBG3 increased the metastatic ability and the protein expression level of the metastasis-associated proteins Snail and Vimentin in low metastatic lung cancer HCC827 cell line. In addition, LNC CRYBG3 contributed to HCC827 cell metastasis in vivo. Mechanistically, LNC CRYBG3 could directly combine with eEF1A1 and promote it to move into the nucleus to enhance the transcription of MDM2. Overexpressed MDM2 combined with MDM2 binding protein (MTBP) to reduce the binding of MTBP with ACTN4 and consequently increased cell migration mediated by ACTN4. In conclusion, the LNC CRYBG3/eEF1A1/MDM2/MTBP axis is a novel signaling pathway regulating tumor metastasis and may be a potential therapeutic target for NSCLC treatment.


Asunto(s)
Proteínas Portadoras/metabolismo , Cristalinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Unión Proteica , ARN Largo no Codificante/genética , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Methods Mol Biol ; 2279: 1-12, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683681

RESUMEN

Due to therapeutic advances, the subclassification of non-small cell lung carcinomas (NSCLC) between the adenocarcinomas and squamous cell carcinomas subtypes is essential for the practice of personalized and targeted medicine. The clinical management for these two NSCLC subtypes is different due to their different molecular properties and histological origins. Immunohistochemistry (IHC) markers such is TTF-1 play a key role in the differentiation of lung adenocarcinomas and squamous cell carcinomas. However, immunohistochemistry is a complex process involving many critical steps and the reliability of results depends on the standardization of the assay as well as the appropriate interpretation. Different laboratories use different reagents and different IHC approaches for the detection of TTF-1 in lung cancer tumors. Here we describe an automated IHC protocol used in our laboratory for the detection of TTF-1 in formalin-fixed, paraffin-embedded (FFPE) tissue sections from lung tumors.


Asunto(s)
Adenocarcinoma del Pulmón , Automatización de Laboratorios , Carcinoma de Células Escamosas , Inmunohistoquímica , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/diagnóstico , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Diagnóstico Diferencial , Femenino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino
8.
Methods Mol Biol ; 2279: 13-21, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683682

RESUMEN

Immunohistochemistry is the technique by which antigens in tissues are detected by means of antigen-antibody reaction. The p40 antibody is directed against the ΔN domain of the ΔNp63 isoform of p63 and is a highly specific marker for the squamous cell carcinoma subtype of non-small cell lung carcinomas (NSCLC). As such, immunohistochemical detection of this antigen in NSCLC biopsies is extremely valuable to assess tumor histological subtype. Herein we describe a manual procedure for performing p40 immunohistochemistry on formalin-fixed paraffin-embedded tissue sections by the indirect polymer-based two-step technique using hydrogen peroxide and 3-3'diaminobenzidine detection system.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Inmunohistoquímica , Neoplasias Pulmonares , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Anticuerpos Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Dominios Proteicos , Isoformas de Proteínas
9.
Methods Mol Biol ; 2279: 23-33, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683683

RESUMEN

Immunohistochemistry (IHC) enables the selective detection of proteins in cells of formalin-fixed-paraffin-embedded (FFPE) tissue sections. This technique plays a key role in the identification and classification of primary lung cancer tumors through the evaluation of the expression of the aspartic proteinase Napsin-A. However, immunohistochemistry is a complex process involving many critical steps and the lack of standardization as well as inappropriate analytical conditions may contribute to inconsistent results between laboratories. Automated immunohistochemistry addresses this issue by ensuring the quality and the reproducibility of the results among different laboratories. Here we describe an automated IHC protocol used in our laboratory for the detection of Napsin-A in FFPE lung tissue sections.


Asunto(s)
Automatización de Laboratorios , Inmunohistoquímica , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Adhesión en Parafina
10.
Methods Mol Biol ; 2279: 35-47, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683684

RESUMEN

Programmed cell death 1 (PD-1) plays an important role in subsiding immune responses, in promoting self-tolerance through suppressing the activity of T-cells, and in promoting differentiation of regulatory T-cells. One of its ligands, programmed cell death ligand 1 (PD-L1) acts as a checkpoint regulator in immune cells and is also expressed in a wide range of cancer types. Anti-PD therapy modulates immune responses at the tumor site, targets tumor-induced immune defects, and repairs ongoing immune responses. Since drugs that target the PD-1/PD-L1 pathways became available as a cancer treatment, there is need for the use of different antibodies to detect the presence of these proteins in tumoral samples by immunohistochemistry or other assays. Because the detection of these antigens in tumor samples is highly clinically informative for guiding treatment decisions, especially to establish the aptness of a patient to receive anti-PD therapy, it is necessary to have a validation process that guaranties that the test results obtained when using antibodies against these proteins are specific, selective, reproducible, and conducive to quantification of antigen abundance in cancer tissue sections. Here we describe an automated immunohistochemistry staining procedure that can be applied for the validation of multiple anti-PD-L1 antibody clones when used for the staining of formalin-fixed, paraffin-embedded lung cancer tissue sections.


Asunto(s)
Automatización de Laboratorios , Antígeno B7-H1/biosíntesis , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Neoplasias Pulmonares , Proteínas de Neoplasias/biosíntesis , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino
11.
Methods Mol Biol ; 2279: 49-57, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683685

RESUMEN

Antibody selection and optimization are crucial to guarantee accurate and reproducible results when using such antibodies for applications such as western blot analysis and immunohistochemistry (IHC). This is especially important when selecting good candidate antibodies that will be used for cancer immunotherapy diagnostics and research. In this chapter, we describe a Western Blot technique as support methodology for the selection and validation of Programmed Cell Death Ligand 1 (PD-L1) antibodies that can be subsequently used in immunohistochemistry applications. Western Blot is a sensitive, specific, and widely available protein characterization technique, used for the detection of specific antigens. PD-L1 is a major immune checkpoint protein that mediates antitumor immune suppression and response, which is routinely detected using IHC in formalin-fixed and paraffin-embedded tissues as part of cancer clinical diagnostic workflows. For this reason, it is critical to define and select the best antibody clones and validate them using different techniques in order to have a reliable detection of positive staining when these antibodies are used in IHC.


Asunto(s)
Antígeno B7-H1/metabolismo , Western Blotting , Inmunohistoquímica , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología
12.
Methods Mol Biol ; 2279: 165-173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683693

RESUMEN

Patient-derived xenografts (PDXs) are created by implanting human tumor tissue or cells into immunodeficent mice, and enable the study of tumor biology, biomarkers and response to therapy in vivo. This chapter describes a method for lung adenocarcinoma (LAC) PDX generation using subcutaneous implantation of tumor tissue and cell suspensions and incorporating the humanization of PDX models by reconstitution with human immune cells.


Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Trasplante de Neoplasias , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Femenino , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID
13.
Methods Mol Biol ; 2279: 175-186, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683694

RESUMEN

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide and is largely inflicted by carcinogens contained in tobacco smoke. The generation of cell lines mimicking traits of human LADC will profoundly advance our understanding of the pathobiology of the disease, as they offer an easy and valuable tool to study the cellular and molecular aspects of carcinogenesis. Here we describe a detailed protocol for the generation of such cell lines, following the exposure of experimental mouse strains to different tobacco carcinogens and isolation of the resulting lung tumors.


Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Fumar/efectos adversos , Fumar/metabolismo , Fumar/patología
14.
Methods Mol Biol ; 2279: 187-198, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683695

RESUMEN

Interrogation and characterization of lung cancer stem cells (CSCs) that are implicated in lung oncogenesis is crucial for our understanding of inter- and intra-tumor heterogeneity and the aggressive nature of the disease, including tumor resistance and relapse in response to conventional therapy. Here, we describe an in vitro surrogate model, namely the "sphere-forming assay," for the derivation, enrichment, and propagation of lung stem/progenitor cells with CSC properties, including self-renewal, tumor initiation capacity and propagation, and differentiation into cells of the tumor bulk, from a murine Kras-mutant lung adenocarcinoma cell line. Self-renewing cancer stem/progenitor cells, in the form of 3D in vitro spheres, can be phenotypically interrogated using downstream techniques such as gene expression analysis (e.g., whole transcriptome sequencing and quantitative real time PCR), which is the focus of this chapter.


Asunto(s)
Adenocarcinoma del Pulmón , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Células Madre Neoplásicas , Transcriptoma , Secuenciación del Exoma Completo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología
15.
Methods Mol Biol ; 2279: 199-212, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683696

RESUMEN

The success of anticancer interventions relies on their ability to ignite an anticancer immune response and to reinstate cancer immunosurveillance. Thus, high dose crizotinib can induce immunogenic cell death (ICD) in cancer cells. If combined with cisplatin, crizotinib sensitizes non-small cell lung cancers (NSCLC) to subsequent (but not simultaneous) immunotherapy with PD-1 immune checkpoint blockade, facilitating the cure of more than 90% of established orthotopic cancers in mice. Here, we detail protocols for the establishment and monitoring of transplantable orthotopic NSCLCs in syngeneic immunocompetent animals. Indeed, TC1 cells establish lung cancer upon their intravenous injection into the tail vein, while Lewis lung carcinoma (LLC) cells can be implanted intrathoracically to generate lung cancers. If transduced with luciferase, both TC1 and LLC cells form tumors that can be conveniently monitored by chemiluminescence. This type of NSCLC model is highly useful for the development of novel curative anticancer therapies.


Asunto(s)
Carcinoma Pulmonar de Lewis , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Trasplante de Neoplasias , Imagen Óptica , Animales , Carcinoma Pulmonar de Lewis/diagnóstico por imagen , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/metabolismo , Ratones
16.
Methods Mol Biol ; 2279: 213-223, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683697

RESUMEN

Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.


Asunto(s)
Anexina A5 , Apoptosis/efectos de los fármacos , Artemisininas/farmacología , Carcinoma de Pulmón de Células no Pequeñas , Fluoresceína-5-Isotiocianato/análogos & derivados , Neoplasias Pulmonares , Propidio/química , Células A549 , Anexina A5/química , Anexina A5/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología
17.
Methods Mol Biol ; 2279: 241-253, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33683699

RESUMEN

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths in the United States. It is extremely difficult to treat, and its survival rate is low. Today, the most effective treatments are still those that implement the platinum anticancer drug cisplatin (CDDP) in combination with other drugs. We previously demonstrated that the naturally occurring compound phenethyl isothiocyanate (PEITC) could be used to sensitize NSCLC cells to CDDP. Furthermore, co-encapsulation of PEITC and CDDP in liposomes enhances their toxicity toward NSCLC cells. We have optimized a liposomal-PEITC-CDDP formulation and investigated its cytotoxicity. We determined that liposomal-PEITC-CDDP is much more toxic toward human NSCLC cell lines than it is toward human normal lung cell lines. In this chapter, we describe detailed methods for preparing liposomal-PEITC-CDDP and determining its cytotoxicity.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Cisplatino , Isotiocianatos , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cisplatino/química , Cisplatino/farmacología , Humanos , Isotiocianatos/química , Isotiocianatos/farmacología , Liposomas , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología
18.
Methods Mol Biol ; 2265: 385-406, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704729

RESUMEN

Metastatic melanoma is one of the most aggressive types of cancers, diffused worldwide and with a significant percentage of lethality. The employment of animal models to test therapeutic strategies against melanoma growth and metastatic spread is of key relevance for cancer biologists. In this regard, the count of metastatic foci in murine lung tissue is one of the recognized methods to monitor macrometastases of melanoma. Here, we illustrate a clonogenic assay method to detect with high sensitivity the presence of single melanoma cells (micrometastases) at the pulmonary level when metastatic foci are still not detectable in the tissue. This method allows for high precision detection and quantification of melanoma metastatic spread to the lung at early stages.


Asunto(s)
Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Ensayo de Tumor de Célula Madre , Animales , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia
19.
Ecotoxicol Environ Saf ; 215: 112127, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33714894

RESUMEN

Numerous epidemiological studies have demonstrated that chronic PM2.5 exposure was associated with the lung carcinogenesis without known potential mechanisms. Exosomes-derived non-coding RNAs, including miRNAs, are proposed to play critical role in the occurrence and development of malignant diseases. So identification of exosomes-derived miRNAs could help us to better understand the molecular toxicity of PM2.5-induced lung cancer. Establishment chronic exposure animal and cell model with PM2.5 was conducted as before. HE staining was used for estimating the histological alternations of lungs in vivo. The expressions of EMT markers in vivo and vitro were quantified by Western blot. Then the exosomes in cell culture supernatant were extracted and the involved miRNAs were extracted and sequenced. The different expression level of miRNAs were verified by RT-PCR. Chronic PM2.5 exposure induced bronchial epithelial cell atypical hyperplasia and massive macrophage infiltration. PM2.5 exposure induce EMT event in vivo and vitro indicated as increased expression of Vimentin and decreased expression of E-cadherin. And five passages of PM2.5 stimulation also induced the release of rich and extractable exosomes in the cell culture supernatant in vitro. Through sequencing, there were differentially expressed 36 miRNAs between PM2.5 chronic exposed and control groups with 1.5-fold and greater differences. Among them, there were 30 exosome-miRNAs upregulated and 6 downregulated expression by PM2.5 exposure. The downregulated expression of miR-29b-2-5p, miR-193b-5p and miR-320c and upregulated expression of miR-100-5p, 125b-5p and unconservative_2_45093 in PM2.5 group were identified and reconfirmed by qRT-PCR. Chronic PM2.5 exposure causes bronchial epithelial cells atypical hyperplasia and induces EMT event in vivo, and it also induce the expression differences of miRNAs in exosome in vitro. Meanwhile, the identified differentially expressed exosome-miRNAs may partially associate with tumorigenesis. To sum up, the identified exosome-miRNAs may play role in the development of lung cancer induced by chronic PM2.5 exposure.


Asunto(s)
MicroARNs/metabolismo , Material Particulado/toxicidad , Animales , Antígenos CD , Cadherinas/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Exosomas , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Regulación hacia Arriba , Vimentina/metabolismo
20.
Life Sci ; 274: 119330, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33711383

RESUMEN

AIMS: The functions and molecular mechanisms of miR-340-3p in lung adenocarcinoma (LUAD) progression remain unclear. On the other hand, the role of HUS1 in LUAD progression should be further explored. MAIN METHODS: Data from cancer database were subjected to bioinformatics analysis. Quantitative real-time PCR and western blot were performed to detect gene expression. Colony formation and MTT assay were performed to examine cell growth in vitro. Wound healing assays and transwell assays were performed to examine cell migration. KEY FINDINGS: Here, our results showed that miR-340-3p was lower expressed in LUAD tissues and LUAD-derived cell lines. And miR-340-3p suppressed the proliferation and migration ability of LUAD cells. Further, miR-340-3p inhibits HUS1 expression, which was higher expressed in LUAD tissues and promoted the proliferation and migration ability of LUAD cells. Moreover, higher HUS1 expression was associated with poor survival rate and shorter survival time in patients with LUAD, and HUS1 expression was negative correlated with that of miR-340-3p in clinical samples. In addition, overexpression of HUS1 counteracted the downregulation of cell growth by miR-340-3p. SIGNIFICANCE: The study mainly indicated that miR-340-3p may play a tumor suppressor role in the progression of LUAD, with the function of restraining HUS1 expression, highlighting a potential therapeutic target for LUAD.


Asunto(s)
Adenocarcinoma del Pulmón/patología , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Pronóstico , Células Tumorales Cultivadas
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