Estudio celular y molecular de los procesos de muerte programada en líneas tumorales sensibles a agentes citostáticos

La apoptosis es una muerte celular programada (MCP) que mantiene el equilibrio entre supervivencia y muerte. Un correcto funcionamiento de las vías apoptóticas contribuye a mantener la integridad genómica mientras que un defecto en la apoptosis puede derivar en el desarrollo de cáncer. Frecuentemente las células tumorales desarrollan mecanismos moleculares con el objetivo de evadir la apoptosis y adquirir resistencia a los agentes citostáticos mediante la sobreexpresión de proteínas antiapoptóticas como Bcl-2 o la mutación de genes supresores de tumores como p53. La alteración en la función de estas proteínas puede derivar en el silenciamiento de los promotores de genes implicados en la reparación del ADN como MGMT. Actualmente la regulación de la apoptosis se considera una diana terapéutica prometedora para el tratamiento del cáncer, lo que ha llevado al desarrollo de agentes citotóxicos cuya función es inducir apoptosis en células tumorales. En este trabajo se presenta un proyecto para analizar in vitro los efectos biológicos a nivel celular y molecular provocados por los agentes citostáticos camptotecina, rituximab, y nivolumab en líneas tumorales de carcinoma de pulmón, H1299, y linfoma no Hodgkin, RL, mediante el uso de técnicas de cuantificación celular (Anexina V-FITC e IP, TUNEL) y el estudio molecular de conversión por bisulfito y PCR de metilación. Se presentan diferentes ensayos dosis-respuesta que permiten estudiar la apoptosis provocada por estos agentes citostáticos y conocer el estado de metilación del promotor de la enzima MGMT. La evaluación molecular y celular ofrece la posibilidad de entender los mecanismos moleculares desarrollados en las células y determinar la dosis óptima para los diferentes tratamientos. Si los resultados obtenidos son prometedores, en el futuro sería conveniente estudiar el efecto sinérgico entre los agentes, ya que esto podría permitir la mejora y desarrollo de tratamientos que puedan llegar a clínic

Cancer-associated fibroblasts modify lung cancer metabolism involving ROS and TGF-β signaling

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contract

SEOM clinical guideline for treatment of kidney cancer (2017)

The goal of this article is to provide recommendations about the management of kidney cancer. Based on pathologic and molecular features, several kidney cancer variants were described. Nephron-sparing techniques are the gold standard of localized disease. After a randomized trial, sunitinib could be considered in adjuvant treatment in high-risk patients. Patients with advanced disease constitute a heterogeneous population. Prognostic classification should be considered. Both sunitinib and pazopanib are the standard options for first-line systemic therapy in advanced renal cell carcinoma. Based on the results of two randomized trials, both nivolumab and cabozantinib should be considered the standard for second and further lines of therapy. Response evaluation for present therapies is a challenge

Concordance between circulating tumor cells and clinical status during follow-up in anaplastic lymphoma kinase (ALK) non-small-cell lung cancer patients

Background: The identification of anaplastic lymphoma kinase (ALK) rearrangements is found in approximately 5% of non-small-cell lung cancers (NSCLCs). However, the development of liquid biopsies as a diagnostic tool is less developed in these cases. This study investigates the use of CTCs during treatment, together with an extended follow-up to correlate with clinical evolution. Patients and Methods: A total of 13 patients out of a cohort of 212 patients with lung adenocarcinoma, presented ALK rearrangements (6%) confirmed by tumor biopsy. A total of 60 serial blood samples were collected from these patients who were prospectively enrolled in the study. Results: All patients had a positive CTC count at baseline (mean = 3). The median follow-up was 9 months (range 1-17 months). Three patients underwent surgery and their CTC counts decreased after the procedure but still remained detectable. After radiotherapy, 3 cases showed an average decrease of 5 CTCs. A total of 6 patients were treated with ALK inhibitors and a partial response was observed in 3 of them, who also presented decreased CTC counts. The other 3 patients presented primary resistance, and their CTC counts were higher than those obtained prior to progression. Conclusion: We believe that the use of CTCs for dynamic monitoring of NSCLC with ALK rearrangement and to detect disease persistence or recurrence may be a reliable technique. CTC counts may also have potential use to monitor the efficacy of ALK inhibitors, facilitating detection of resistance to treatmentThis study was supported by Carlos III Institute of Health, Spanish Ministry of Science and Innovation, and European Regional Development Fund (grant number: PI16/01818 and PIE14/00064), D. Pérez-Callejo is supported by SEOM-Río-Hortega contract, A Romero is supported by Joan Rodés fellowship (grant number: JR14/00017) and M Sánchez-Beato is supported by Miguel Servet contract (CP11/00018 and CPII16/00024

Experience with Sunitinib in metastatic renal cell carcinoma (mRCC) patients: pooled analysis from 3 Spanish observational prospective studies

[Abstract] Background: A pivotal, randomized, phase III trial demonstrated a statistically significant superiority of sunitinib over interferon-α in metastatic renal cell carcinoma (mRCC) patients. Objective: To evaluate the effectiveness and safety of sunitinib in patients with advanced or mRCC in routine clinical practice. Methods: Retrospective pooled analysis of clinical data from three observational and prospective studies carried out between 2007 and 2011 in 33 Spanish hospitals. Tumor response, Progression-free survival (PFS) and overall survival (OS), and main sunitinib-related toxicities were registered. Results: 224 patients were analyzed. Median PFS 10.6 months (95% CI: 9.02–12.25), median OS 21.9 months (95% CI: 17.2–26.6). Objective response rate (ORR) 43.8% (95% CI: 36.8–50.7). Median time to PR was 3.8 months (95% CI: 3.86–5.99) and to CR 8.2 months (95% CI: 4.75–9.77). The most common ≥ grade-3 AEs were asthenia/fatigue (18.7%), hand-foot syndrome (6.2%), hypertension (5.8%) and neutropenia (4.8%). Hand-foot syndrome, diarrhea and mucositis were confirmed as independent predictors for PFS and/or OS in a multivariate analysis (p < 0.05) Conclusions: Outcomes with sunitinib in daily clinical practice resemble those obtained in clinical trials. Long-term benefit with sunitinib is possible in advanced RCC patients but the appropriate management of toxicities is mandatory to enable patients to remain on treatment

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition

Background: Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. Methods: Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). Results: Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles’ heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. Conclusion: These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contrac

Apoptosis y criopreservación de progenitores hematopoyéticos de sangre periférica

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica. Fecha de lectura: 27-06-2002La cantidad de células CD34+ infundidas constituye un parámetro de gran valor para asegurar el 6xito del implante del producto de células progenitoras hematopoy6ticas trasplantado. Su cuanticación por citometría de flujo es considerado hoy el método de elección aceptado para la determinación de PHSP. Este trabajo propone el desarrollo de una técnica citom6tnca nueva que incluye todas las premisas conocidas para la cuantificación de cblulas CD34+ en muestras de leucoaféresis y que permite conocer además, su viabilidad mediante identikación y cuantiiición de las dlulas CD34+ apoptóticas y necróticas. Para comprobar la validez del método propuesto, las determinaciones se realizaron en comparación con dos métodos ya estandarizados, y en dos momentos diferentes: antes de la congelación y tras la descongelación de los progenitores hematopoybticos. Además, se estudia el efecto de la criopresewación sobre las células mediante la incorporación del marcador de detección de apoptosis precoz Anexina V. Los resultados obtenidos indican que con nuestro mbtodo es mayor la población de progenitores que se obtiene tanto en la precongelación como tras la descongelación. Ello es debido a la aplicación del sistema de ventanas consecutivas, la utilización de fluoroesferas que permite la cuanticación de la concentración celular, y a la utilización de fluoroar>mos que ofrecen marcaje más específico y mayor avidez por el antígeno CD34. En cuanto al estudio del efecto de la criopresewación se detecta disminución de la concentración de progenitores tras la descongelación. Estas variaciones sin embargo, no parecen estar relacionadas con p6rdida selectiva de células sino con la disminución en la inmunorreactividad por alteración del antígeno CD34. Finalmente, el estudio de otros factores como la utilización de determinada quimioterapia en el tratamiento previo, el uso de quimioterapia en la movilización de los progenitores y el número de aféresis consecutivas, sí parecen influir en alguna medida sobre la viabilidad de las células hematopoybticas CD34+. Por todo ello, se considera que la aplicación de esta metodologia es de gran interés clínico y que su estandarización puede conducir a obtener un producto mejor para su utilización en el trasplante.The amount of CD34+ cells infused is considered the best parameter for predicting the success of engraftment after hematopoietic progenitors cell transplantation. Nowadays, the flow cytometric assays are widely accepted for quantication of CD34+ cells. This work propose the development of a new cytometric technique that, including al1 the premises for the quatiication of CD34+ cells in leucaphereais sampies, permit to know it viabili by the detection and quanüficatii of CD34+ apoptotics and necrotics cells. To verify the validity of the proposed method, the determination of the progenitors cells was performed in cornparation with two standarized methods and at two dirent times: at the end uf harvesting before aiopreservation and after thawing. Furthemiore was studi the criopreservatim efiects on the cells by staining with apoptosis marker Anexina V. The results indicate that our method, the mean percentage of CD34+ cells obtained is bigger than other methods for two moments analysed. This is due to the application of the consewtive window, Boolean system. the use of fluorospheres, singleplafform assays that pernil kmm cell concentration. and the use of fluorocnmies that ofíers a markers more specific and b i i r a v a i for the CD34 antigen. According to the study of the aiopreservation effects, is detected a decrease of the concentration of progenitors after thawing. These variations however do not seem to be related with the selective ieakage of cells but with readivity immune decrease by the alterati of CD34 entigen. Finally, the study of other factors like the use of a specifi quirniotherapy in the previous treatment, the mobilization regimen and the number of consecutive apheresis, appears to affed on the viabili of CD34+ hematopoietim cells. For al1 of this, is considered that the application of this methodology is of a great dinic intmst and that it standardisation can drive to have a better product to use in transplant

PGC-1alpha levels correlate with survival in patients with stage III NSCLC and may define a new biomarker to metabolism-targeted therapy

Lung cancer remains the leading cause of cancer-related death worldwide, with one-third diagnosed with locally advanced (stage III) disease. Preoperative induction chemo-radiotherapy is key for the treatment of these patients, however conventional cisplatin based approaches has apparently reached a plateau of effectiveness. In the search for new therapies, the targeting of tumor metabolism is revealed as an interesting option to improve the patient's responses. Here we describe the importance of PGC-1alpha and GAPDH/MT-CO1 ratio levels as surrogates of the Warburg effect from a series of 28 stage III NSCLC patients, on PFS, OS and PET uptake. Moreover, our results show a great variability between tumors of different individuals, ranging from very glycolytic to more OXPHOS-dependent tumors, which compromises the success of therapies directed to metabolism. In this sense, using 3 different cell lines, we describe the relevance of Warburg effect on the response to metabolism-targeted therapies. Specifically, we show that the inhibitory effect of metformin on cell viability depends on cell's dependence on the OXPHOS system. The results on cell lines, together with the results of PGC-1alpha and GAPDH/MT-CO1 as biomarkers on patient's biopsies, would point out what type of patients would benefit more from the use of these drugs.Work in the authors’ laboratories is supported by “Instituto de Salud Carlos III” PI13/01806 and PIE14/0064 to MP. RLB is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contract.Peer reviewe

Radiobiological Effects Induced by X-ray (LINAC) Irradiation: Experiments and Modelling

Part of the Bioanalysis book series (BIOANALYSIS, volume 8)In this study we present a modelling procedure that combines the standard electromagnetic physics of Geant4 for photons with the LEPTS event by event Monte Carlo simulation programme for secondary electrons. LEPTS provides detailed information of the energy deposition and the type and number of collision events taking place in a 1 m3 liquid water phantom when irradiated with 6 MV photons generated by a LINAC accelerator. The simulation is compared to a radiobiological experiment determining the biological effects (cell cycle alteration, early apoptosis, DNA damage) induced by the 6 MeV CLINAC radiation to a Jurkat T lymphocyte culture in a water phantom. Notable cell cycle alterations were found between 24 and 48 h after a 2 Gy radiation dose. Early apoptosis and DNA fragmentation has been analysed at 48, 72 and 96 h post irradiation. As expected, the sequence of the apoptotic process has been clear in the irradiated area up to 48 h after radiation. However, substantial apoptotic cells have also been identified outside of this area, in the penumbra and even in the low dose area, where a maximum in the percentage of DNA fragmentation is shown. We attributed this effect to the fact that although the average energy of primary and secondary particles in these areas is lower than those in the irradiated area, their interaction cross sections are much higher, thereby increasing the probability of producing damage.This study has been mainly supported by the EU through the FP7-PEOPLE-2013-ITN programme (project ARGENT-608163). It has also been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (project FIS2016-80440). We also acknowledge the Hospital Universitario Puerta de Hierro of Madrid for providing the required equipment and services to perform the presented experiments