Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines

We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.This research was funded by a Grant from Instituto de Salud Carlos III Grant PI012/02025 co-supported by FEDER funds and PRECIPITA crowdfunding platform from Fundación Española para la Ciencia y la Tecnología (Fecyt) to M. Saceda and AMACMED (Asociación de mujeres afectadas por cáncer de mama de Elche y Comarca) and Monica Moraleda donation to M. Saceda. The Spanish Ministry of Economy and Competitiveness (MINECO, Project RTI2018-096724-B-C21) and the Generalitat Valenciana (PROMETEO/2016/006) supported the work in the Encinar laboratory

The intrinsically disordered, epigenetic factor RYBP binds to the citrullinating enzyme PADI4 in cancer cells

14 p.-6 fig.-1 tab.RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 μM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.This research was funded by Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033/ and “ERDF A way of Making Europe” [PID2021-127296OB-I00 to AVC; and PDC2022-133952-I00 to EF]; by Instituto de Salud Carlos III co-funded by European Social Fund “Investing in your future” [CP19/00095 to CdJ] [PI22/00824 to MS and CdJ] [PI18/00394 to OA]; by Diputación General de Aragón [“Protein targets and Bioactive Compounds group” E45-20R to AVC, and “Digestive Pathology Group” B25-20R to OA], and by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana) [CAICO 2021/0135 to CdJ and JLN].Peer reviewe

Combinación de una terapia enzimática con nanotecnología para el tratamiento del cáncer

La terapia enzimática suicida consiste en dirigir una enzima hacia el tumor y, a continuación, administrar de forma sistémica un profármaco inocuo que debe ser sustrato de la enzima y que, al ser procesado por ella, se convertirá en un fármaco anticancerígeno activo. En este trabajo se ha evaluado la eficacia del tratamiento con la enzima Daminoácido oxidasa (DAO) de Rhodotorula gracilis junto con D-alanina frente a líneas celulares de carcinoma de páncreas, carcinoma colorrectal y glioblastoma. La DAO cataliza la oxidación de D-aminoácidos, convirtiéndolos en alfa-cetoácidos y amonio y generando H2O2, que induce estrés oxidativo. Concretamente, se ha utilizado una quimera que presenta la DAO unida al domino de unión a colina de la amidasa N-acetilmuramoil-Lalanina (CLytA) de Streptococcus pneumoniae, permitiendo la inmovilización de la enzima en soportes que presenten colina o derivados, como el dietilaminoetanol (DEAE). Los resultados de este estudio demuestran que CLytA-DAO induce un efecto antiproliferativo en la mayoría de las líneas celulares de carcinoma de páncreas, carcinoma colorrectal y glioblastoma utilizadas. Esta disminución de la proliferación celular se debe a un efecto citotóxico causado por el aumento de especies reactivas de oxígeno en el interior celular, que induce daños en el ADN y en la membrana plasmática, así como la disminución del potencial de membrana mitocondrial. Al analizar el efecto de la enzima en células no tumorales, se observó que no se producía muerte celular o bien ésta era significativamente inferior a la observada en las líneas tumorales. La utilización de la DAO quimérica permitió además su inmovilización en nanopartículas magnéticas funcionalizadas con DEAE, lo que aumentó el efecto del tratamiento al mejorar la estabilidad de la enzima a 37°C. Se estudió también la inmovilización en nanopartículas de oro y cápsulas de alginato, aunque el efecto citotóxico disminuyó considerablemente con respecto al observado con la enzima libre. Al analizar el mecanismo de muerte celular, se observó que el tratamiento con CLytA-DAO y D-Ala es capaz de activar diferentes tipos de muerte celular, de modo que en las líneas celulares de carcinoma de páncreas y colon se produce una necrosis regulada mientras que en las de glioblastoma se activa la vía intrínseca de la apoptosis. Entre las diferentes líneas celulares estudiadas hubo dos resistentes al efecto citotóxico inducido por CLytA-DAO, Hs766T de carcinoma de páncreas y HT-29 de carcinoma colorrectal. Al profundizar en el mecanismo de resistencia de ambas líneas celulares, se observó que está relacionado con la sobreexpresión de genes involucrados en mecanismos de detoxificación, en la respuesta frente al estrés oxidativo y en la supervivencia celular. La expresión de estos genes fue estudiada también en biopsias de pacientes de los tres tipos de tumores y, los resultados sugieren que el tratamiento con CLytA-DAO y D-Ala podría ser eficaz en un alto porcentaje de pacientes. Se demostró que la enzima CLytA-DAO también podría ser utilizada en combinación con radioterapia, quimioterapia, o con tratamientos epigenéticos o basados en reducir la reparación del ADN para potenciar el efecto sobre los pacientes. En conclusión, los resultados de este trabajo indican que el tratamiento con CLytA-DAO y D-Ala muestra un gran potencial como estrategia terapéutica anticancerígena, tanto por sí misma como en combinación con otros tratamientos, en pacientes con cáncer de páncreas, colorrectal y glioblastoma.Suicide enzyme therapy consists of targeting an enzyme to the tumor and then, to administer systemically a harmless prodrug that must be a substrate for the enzyme and that it will become an active anticancer drug after reacting with it. In this work, treatment efficacy with the enzyme D-amino acid oxidase (DAAO) from Rhodotorula gracilis together with D-alanine against pancreatic carcinoma, colorectal carcinoma and glioblastoma cell lines has been evaluated. DAAO catalyzes D-amino acids’ oxidation, converting them into alpha-ketoacids and ammonia and generating H2O2, which induces oxidative stress. Specifically, a chimera has been used, which presents DAAO bound to the choline-binding domain of the N-acetylmuramoyl-L-alanine amidase (CLytA) from Streptococcus pneumoniae, allowing the enzyme immobilization on supports containing choline or derivatives, such as diethylaminoethanol (DEAE). The results of this study demonstrate that CLytA-DAAO induces an antiproliferative effect in most of the pancreatic carcinoma, colorectal carcinoma and glioblastoma cell lines used. This decrease in cell proliferation is due to a cytotoxic effect caused by the increase in reactive oxygen species inside the cell, which induces damage to DNA and plasma membrane as well as a decrease in the mitochondrial membrane potential. By analyzing the enzyme effect in non-tumor cells, absence or significantly lower levels of cell death were observed in comparison to tumor cell lines. In addition, the use of a chimeric DAAO allowed its immobilization on DEAE-functionalized magnetic nanoparticles, increasing the effect of the treatment by improving the enzyme stability at 37°C. Immobilization in gold nanoparticles and alginate capsules was also studied, although the cytotoxic effect decreased considerably compared to that observed with the free enzyme. Analysis of the cell death mechanisms showed that treatment with CLytA-DAAO and D-Ala triggers different types of cell death. In pancreatic and colorectal carcinoma cell lines a regulated necrosis occurs while in glioblastoma cell lines the intrinsic pathway of apoptosis is activated. Among the different cell lines studied, two of them were resistant to the cytotoxic effect induced by CLytA-DAAO, Hs766T from pancreatic carcinoma and HT-29 from colorectal carcinoma. By deepening in the mechanism of resistance in both cell lines, it was observed that it is related to the overexpression of genes involved in detoxification mechanisms, response to oxidative stress and cell survival. The expression of these genes was also studied in biopsies from patients with each of the three types of tumors, and the results suggest that treatment with CLytA-DAAO and D-Ala could be effective in a high percentage of these patients. In addition, it was shown that the CLytADAAO enzyme could also be used in combination with either radiotherapy, chemotherapy, epigenetic therapy, or therapy based on reducing DNA repair, in order to enhance the effect on patients. In conclusion, the results of this work indicate that treatment with CLytA-DAAO and D-Ala shows a high potential as an anticancer therapeutic strategy, both by itself and in combination with other treatments, in patients with pancreatic and colorectal carcinoma and glioblastoma

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines

We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.This research was funded by a Grant from Instituto de Salud Carlos III Grant PI012/02025 co-supported by FEDER funds and PRECIPITA crowdfunding platform from Fundación Española para la Ciencia y la Tecnología (Fecyt) to M. Saceda and AMACMED (Asociación de mujeres afectadas por cáncer de mama de Elche y Comarca) and Monica Moraleda donation to M. Saceda. The Spanish Ministry of Economy and Competitiveness (MINECO, Project RTI2018-096724-B-C21) and the Generalitat Valenciana (PROMETEO/2016/006) supported the work in the Encinar laboratory

Cell death mechanisms induced by CLytA-DAAO chimeric enzyme in human tumor cell lines

23 p.-11 fig.The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer.This research was funded by Spanish Instituto de Salud Carlos III, grant number PI01202025, and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to M.S., Miguel Servet Program from Instituto de Salud Carlos III (CP19/00095) to C.d.J.R. and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to J.M.S. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).Peer reviewe

CLytA-DAAO, free and immobilized in magnetic nanoparticles, induces cell death in human cancer cells

19 p.-8 fig.-1 tab.D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.This research was funded by Spanish Instituto de Salud Carlos III, grant number PI01202025, and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to MS and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to JMS. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).Peer reviewe

New therapy for pancreatic cancer based on extracellular vesicles

Pancreatic Ductal Adenocarcinoma (PDAC), is the most common aggressive cancer of the pancreas. The standard care of PDAC includes tumor resection and chemotherapy, but the lack of early diagnosis and the limited response to the treatment worsens the patient’s condition. In order to improve the efficiency of chemotherapy, we look for more efficient systems of drug delivery. We isolated and fully characterized small Extracellular Vesicles (EVs) from the RWP-1 cell line. Our study indicates that the direct incubation method was the most efficient loading protocol and that a minimum total amount of drug triggers an effect on tumor cells. Therefore, we loaded the small EVs with two chemotherapeutic drugs (Temozolomide and EPZ015666) by direct incubation method and the amount of drug loaded was measured by high-performance liquid chromatography (HPLC). Finally, we tested their antiproliferative effect on different cancer cell lines. Moreover, the system is highly dependent on the drug structure and therefore RWP-1 small EVsTMZ were more efficient than RWP-1 small EVsEPZ015666. RWP-1 derived small EVs represent a promising drug delivery tool that can be further investigated in preclinical studies and its combination with PRMT5 inhibitor can be potentially developed in clinical trials for the treatment of PDAC

CLytA-DAAO chimeric enzyme bound to magnetic nanoparticles. A new therapeutical approach for cancer patients?

24 p.-10 fig.-3 tab.D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma.This research was funded by Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO), grant number UGP-19-063, and co-supported by PRECIPITA crowdfunding platform from Spanish Foundation for Science and Technology (FECYT) and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to M.S., Miguel Servet Program from Instituto de Salud Carlos III (CP19/00095) to C.d.J.R. and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to J.M.S. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).Peer reviewe

Coneixement del cànon artístic del còmic a les aules universitāries: possibilitats didāctiques. Anàlisi comparada de diverses universitats

Aquesta memòria reflecteix la faena de la xarxa 4698 sobre el coneixement del cànon artístic del còmic entre l’alumnat universitari. S’hi explica l’elaboració i difusió d’una enquesta respecte un llistat de trenta obres compilat pel grup Unicòmic de la Universitat d’Alacant a partir de publicacions especialitzades, i amb la qual es pretén saber quin és el grau de reconeixement d’historietes clàssiques. Inicialment, la pretensió era que aquesta enquesta s’estenguera a facultats diverses de la UA i a altres universitats, per la qual cosa els components de la xarxa pertanyen a institucions acadèmiques diverses. No obstant això, les dificultats derivades del confinament provocaren que l’enquesta se centrara en alumnat de la Facultat d’Educació; així mateix, la supervisió per a poder aconseguir un espectre major d’enquestats no fou tan efectiva com s’havia previst. En tot cas, aquest projecte representa un pas avant en un procés d’investigació que, després d’altres xarxes docents en aquesta universitat mateix i en les quals s’ha anat compilant un corpus bibliogràfic al voltant del cànon artístic del còmic, ara ens permet avançar vers la confrontació amb els interessos de l’alumnat per a poder implementar-hi en el futur un cànon segons nivells educatius i amb propostes pedagògiques pertinents.Sin financiaciónNo data 2020UE