[ES] La presente tesis doctoral titulada "La terapia senolítica como nuevo enfoque terapéutico para la prevención de la cardiotoxicidad inducida por la doxorrubicina" se centra en explorar la eliminación de células senescentes (senolisis) in vitro e in vivo mediante la administración del fármaco senolítico, navitoclax, como opción terapéutica para prevenir la cardiotoxicidad asociada al tratamiento antitumoral con doxorrubicina. La exposición a doxorrubicina afecta gravemente a la población de células cardíacas tanto en ratones como en corazones humanos al inducir senescencia prematura y navitoclax es uno de los senolíticos más potentes y ampliamente conocido en el campo de la senescencia celular pero su administración está asociada a la aparición de efectos adversos, principalmente trombocitopenia. Con el objetivo de mejorar el perfil terapéutico del fármaco, se han desarrollado dos estrategias específicamente dirigidas a células senescentes que incluyen la encapsulación y el desarrollo de un profármaco basado en navitoclax. En la primera sección experimental, abordamos la inducción de senescencia en un modelo in vitro de cardiomiocitos y evaluamos la terapia senolítica como estrategia terapéutica. Desarrollamos y caracterizamos, así mismo, las nanopartículas y la prodroga basada en navitoclax. Este nanodispositivo senolítico se basa en MSNs cargadas con navitoclax y funcionalizadas con un hexa-galacto-oligosacárido (galactán). Por su parte, la prodroga se obtiene tras la conjugación del navitoclax a una molécula de galactosa acetilada. En ambos casos, el mecanismo de la terapia dirigida radica en la presencia de la enzima lisosomal ß-galactosidasa, la cual presenta mayor expresión en las células senescentes. Al entrar en el lisosoma de células senescentes, los enlaces glicosídicos se hidrolizan por acción de la enzima, liberando el fármaco para su función. Los resultados muestran un aumento del efecto terapéutico del fármaco libre. A continuación, ponemos a punto un modelo murino de cardiotoxicidad donde demostramos que la administración sistémica de doxorrubicina induce la expresión de marcadores de cardiotoxicidad y senescencia en el corazón de ratones tratados y contribuye al deterioro de la función cardíaca de los animales seguido por ecografía. En este modelo preclínico, el tratamiento combinado de doxorrubicina con el senolítico navitoclax, en las diferentes formulaciones descritas, conduce a la disminución significativa de los marcadores de senescencia y cardiotoxicidad junto con el restablecimiento de la función cardíaca y, se observa, de manera similar con las tres estrategias mencionadas: fármaco libre, fármaco encapsulado y fármaco en formulación de prodroga. Los resultados científicos presentados en esta tesis resaltan el papel de la senescencia en la progresión de cardiotoxicidad por la administración de doxorrubicina y se concluye que los sistemas senolíticos aquí evaluados podrían ser una herramienta importante para el desarrollo de nuevas estrategias terapéuticas en el campo de la prevención de efectos secundarios asociados a terapia. / [CA] La present tesi doctoral titulada "La teràpia senolítica com a nou enfocament terapèutic per a la prevenció de la cardiotoxicitat induïda per la doxorrubicina" se centra en explorar l'eliminació de cèl·lules senescents (senolisis) in vitro i in vivo mitjançant l'administració del fàrmac senolític, navitoclax, com a opció terapèutica per a previndre la cardiotoxicitat associada al tractament antitumoral amb doxorrubicina. L'exposició a doxorrubicina afecta greument a la població de cèl·lules cardíaques tant en ratolins com en cors humans en induir senescència prematura y navitoclax és un dels senolítics més potents i àmpliament conegut en el camp de la senescència cel·lular però la seua administració està associada a l'aparició d'efectes adversos, principalment trombocitopenia. Amb l'objectiu de millorar el perfil terapèutic del fàrmac, s'han desenvolupat dues estratègies específicament dirigides a cèl·lules senescents que inclouen l'encapsulació i el desenvolupament d'un profàrmac basat en navitoclax. En la primera secció experimental, abordem la inducció de senescència en un model in vitro de cardiomiòcits i avaluem la teràpia senolítica com a estratègia terapèutica. Desenvolupem i caracteritzem, així mateix, les nanopartícules i la prodroga basada en navitoclax. Aquest nanodispositiu senolític es basa en MSNs carregades amb navitoclax i modificades amb un hexa-galacto-oligosacàrid (galactán). Per part seua, la prodroga s'obté després de la conjugació del navitoclax a una molècula de galactosa acetilada. En tots dos casos, el mecanisme de la teràpia dirigida radica en la presència de l'enzim lisosòmic ß-galactosidasa, la qual presenta major expressió en les cèl·lules senescents. En entrar en el lisosoma de cèl·lules senescents, els enllaços glicosídics s'hidrolitzen per acció de l'enzim, alliberant el fàrmac per a la seua funció. Els resultats mostren un augment de l'efecte terapèutic del fàrmac lliure. A continuació, posem a punt un model de ratolí on vam demostrar que l'administració sistèmica de doxorrubicina indueix l'expressió de marcadors de cardiotoxicitat i senescència en el cor de ratolins tractats i contribueix a la deterioració de la funció cardíaca dels animals seguit per ecografia. En aquest model preclínic, el tractament combinat de doxorrubicina amb el senolític navitoclax, en les diferents formulacions descrites, condueix a la disminució significativa dels marcadors de senescència i cardiotoxicitat juntament amb el restabliment de la funció cardíaca i, s'observa, de manera similar amb les tres estratègies esmentades: fàrmac lliure, fàrmac encapsulat i fàrmac en formulació de prodroga. Els resultats científics presentats en aquesta tesi ressalten el paper de la senescència en la progressió de cardiotoxicitat per l'administració de doxorrubicina i es conclou que els sistemes senolítics ací avaluats podrien ser una eina important per al desenvolupament de noves estratègies terapèutiques en el camp de la prevenció d'efectes secundaris associats a teràpia. / [EN] The present PhD thesis entitled " Pharmacological senolysis as a new therapeutic approach for the prevention of doxorubicin-induced cardiotoxicity" focuses on exploring the elimination of senescent cells (senolysis) in vitro and in vivo by administration of the senolytic drug, navitoclax, as a therapeutic option to prevent cardiotoxicity associated with antitumor treatment with doxorubicin. Doxorubicin exposure severely affects the cardiac cell population in both mouse and human hearts by inducing premature senescence and navitoclax is one of the most potent and widely known senolytics in the field of cellular senescence, but its administration is associated with the occurrence of adverse effects, mainly thrombocytopenia. In the first experimental section, we addressed the induction of senescence in an in vitro cardiomyocyte model and evaluated senolytic therapy as a therapeutic strategy. We also developed and characterized the nanoparticles targeting senescent cells and the navitoclax-based prodrug. The senolytic nanodevice is based on MSNs loaded with navitoclax and functionalized with a hexa-galacto-oligosaccharide (galactan). The prodrug is obtained after conjugation of navitoclax to an acetylated galactose molecule. In both cases, the mechanism of targeted therapy lies in the presence of the lysosomal enzyme ß-galactosidase, which is highly expressed in senescent cells. Upon entering the lysosome of senescent cells, the glycosidic bonds are hydrolyzed by the action of the enzyme, releasing the drug. The results show an increase in the therapeutic effect of the free drug. Next, we developed a murine model of cardiotoxicity in which we demonstrated that systemic administration of doxorubicin induces the expression of markers of cardiotoxicity and senescence in the heart of treated mice and contributes to the deterioration of the cardiac function of the animals followed by echocardiography. In this preclinical model, the combined treatment of doxorubicin with the senolytic navitoclax, in the different formulations described above, leads to significant decrease in senescence and cardiotoxicity markers along with restoration of cardiac function. Similar results were observed with the three strategies mentioned: free drug, encapsulated drug, and prodrug formulation. The scientific results presented in this thesis highlight the role of senescence in the progression of cardiotoxicity by doxorubicin administration and it is concluded that the senolytic systems evaluated here could be an important tool for the development of new therapeutic strategies in the field of prevention of therapy-associated side effects and represent an alternative to the limitations of current treatments. / Lérida Viso, A. (2023). Pharmacological Senolysis as a New Therapeutic Approach for the Prevention of Doxorubicin-Induced Cardiotoxicity [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194709

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Die Überlebensrate von Patienten mit duktalem Adenokarzinom des Pankreas ist sowohl in primär resektablen als auch im lokal fortgeschrittenen und metastasierten Stadium kurz. Das duktale Adenokarzinom des Pankreas breitet sich rasch aus und wird aufgrund fehlender Frühsymptome oft erst im fortgeschrittenen Stadium diagnostiziert. Gegenwärtig fehlen spezifische Tumormarker, die eine frühzeitige Diagnose erlauben würden. Aufgrund zahlreicher Mechanismen der primären und sekundären Chemoresistenz ist das Pankreaskarzinom verhältnismäßig resistent gegenüber konventioneller systemisch verabreichter Chemotherapie, antikörperbasierten sowie niedermolekularen Therapiestrategien, Enzyminhibitoren, Bestrahlung und chirurgischer Therapie. Ein vielversprechender Angriffspunkt zur zielgerichteten Therapie des Pankreaskarzinoms eröffnet die tumorspezifische Expression des GnRH-I Rezeptors. In dieser Arbeit konnte der GnRH-IRezeptor durch RT-PCR und immunhistochemisch in 32,5% der duktalen Adenokarzinome des Pankreas nachgewiesen werden. Es wurde gezeigt, dass die Behandlung mit dem Hybridwirksoff AESZ-108 (AN-152), einem zytotoxischen GnRH-Analogon in vitro und in vivo Apoptose in den GnRH-IRezeptor- positiven Pankreaskarzinomzelllinien induziert. Apoptose wurde durch den intrinsischen Signalweg über den Zusammenbruch des mitochondrialen Membranpotentials vermittelt und führte zu DNA-Fragmentierung des Nukleus wie fluoreszenzmikroskopisch gezeigt werden konnte. Das zytotoxische GnRH-Analogon AESZ-108 (AN-152) führt in vivo zu einer signifikanten Inhibition des Tumorwachstums im Vergleich zur Therapie mit dem Anthrazyklin Doxorubicin, welches zu keiner signifikanten Inhibition des Pankreaskarzinomwachstums führt. Die rezeptorvermittelte Aufnahme ermöglicht eine selektive Therapie. Nach rezeptorvermittelter Endozytose wird das an die D-Lys6-Seitenkette gebundene Doxorubicin spezifisch im Nukleus der rezeptorpositiven Karzinomzellen freigesetzt. Die Ergebnisse dieser Arbeiten zeigen, dass AESZ-108 (AN-152) ein geeigneter Ansatz zur selektiven Chemotherapie GnRH-I-Rezeptor positiver humaner duktaler Adenokazinome des Pankreas ist.

Over one in five cancer patients will develop brain metastases and prognosis remains poor. Effective chemotherapeutics for primary systemic tumours have limited access to brain metastases owing to the blood-brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilising the BBB at sites of cerebral metastases. Tumour necrosis factor was injected intravenously into mouse models of haematogenously induced brain metastasis. BBB permeability was assessed through histology and in vivo MRI and SPECT. Tumour burden and neuroinflammation were assessed after injection of TNF with Caelyx or a novel therapeutic. Mechanism of permeabilisation was investigated through histology and receptor-specific agonist antibodies. Administration of TNF dose-dependently permeabilised the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect after six hours. Metastasis-specific uptake of radiolabelled trastuzumab was also demonstrated following systemic cytokine administration. Administration of liposomal doxorubicin formulations in conjunction with TNF reduced tumour burden and mean metastasis size. Localised expression of TNFR1 was evident on the vascular endothelium associated with brain metastases. Human brain metastases displayed a similar TNF receptor profile compared to the mouse model. These findings describe a new approach to selectively permeabilise the BBB at sites of brain metastases, thereby enabling detection of currently invisible micrometastases and facilitating tumour-specific access of chemotherapeutic agents. We hypothesize that this permeabilisation works primarily though TNFR1 activation and, owing to the similar TNFR1 expression profiles in mouse models and human condition, the strategy has the potential for clinical translation.

Master of Science / Department of Clinical Sciences / Mary Lynn Higginbotham / Objectives – To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSCs) in culture, to determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSCs and canine osteosarcoma (OSA) D17 cell viability, and to determine the effects of conditioned media from WJMSCs loaded with micellar nanoparticles containing DOX on OSA D17 cell viability. Sample Population – Canine WJMSCs containing various concentrations of DOX micelles and canine OSA D17 cells. Procedures – WJMSCs were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to culture plates containing canine OSA D17 cells to determine micelle effects on cell growth and viability. Conditioned media from culture plates containing canine WJMSCs incubated with various DOX micelle concentrations was added to OSA D17 cells for conditioned media experiments. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess OSA D17 cell viability. A trypan blue stain was also utilized to perform cell counts to determine the effect of the DOX micelles on stromal cell growth. Results – WJMSCs were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased OSA D17 cell viability. OSA D17 cell viability was also decreased following incubation with conditioned media from canine WJMSCs loaded with micellar nanoparticles containing DOX. Significant decreases with the conditioned media of canine WJMSCs loaded with 10μM micelles occurred at 48 hours (p < 0.005) and at 72 and 96 hours (p < 0.0001). Significant decreases were also observed with the 1 μM DOX micelles at 72 hours (p < 0.005) and 96 hours (p < 0.0001). WJMSC numbers decreased in a dose dependent manner following incubation with DOX micelles. Changes in WJMSC number was not caused by increased cell death as all variables produced similar percentages of dead cells. Conclusions – Canine WJMSCs were successfully isolated and maintained in culture. Stromal cells containing DOX micellar nanoparticles induced OSA D17 cell cytotoxicity while inducing an anti-proliferative, rather than cytotoxic effect, on the WJMSC. These data support future in vivo experiments utilizing canine WJMSCs and micellar nanoparticles.

vi ABSTRACT IL-2 has been used in cancer therapy and also for other applications like treatment of chronic viral infections or as an adjuvant for vaccines. However, treatment with IL-2 is rather difficult due to its severe side effects. These toxicities, associated with high-dose treatment necessary for IL-2 to function, have been found the most limiting factor for IL- 2 applications. Further, particular anti-IL-2 monoclonal antibodies (mAb) can actually increase biological activity of IL-2 rather than block it. Binding of IL-2 to anti-IL-2 mAb creates a superagonistic immunocomplexes which have dramatically higher and selective biological activity in comparison to free IL-2 in vivo. Such approach may finally over- come the difficulties associated with administration of IL-2, thus opening brand new scopes for IL-2 and its application not only in the field of tumor therapy. We have shown that IL-2 immunocomplexes composed of IL-2 and anti-IL-2 mAb S4B6 (IL-2/S4B6) stimulate predominantly cells expressing CD122 and CD132 (dimeric IL-2 receptor), i.e. NK and MP CD8+ T cells, with Treg,  T and NKT cells being expanded as well. IL-2/S4B6 are able to drive the expansion of activated naive CD8+ T cells into functional memory-like CD8+ T cells. Moreover, these immunocomplexes exert therapeu- tical potential alone...

O estado de metilação é suscetível a mudanças quando os organismos são expostos a agentes ambientais tais como componentes dos alimentos e medicamentos. Uma dieta rica em metionina (Met) poderia modular a concentração de S-adenosilmetionina (SAM) e S-adenosilhomocisteína (SAH) e alterar o estado de metilação da região promotora de genes supressores de tumores. Tanto a hipometilação global quanto a hipermetilação de genes específicos estão envolvidas na instabilidade genômica e poderiam resultar em dano ao DNA. Este estudo avalia se a dieta suplementada com Met associada a doxorrubicina (DXR), um fármaco antitumoral que induz espécies reativas, resulta em alterações no estado de metilação da região promotora do gene TP53, na razão SAM/SAH, na concentração de glutationa (GSH) e em dano ao DNA. Quarenta ratos machos Wistar foram separados em dois grupos: dieta suplementada com Met (ração comercial acrescida de 2% Met) e dieta controle (ração comercial) por seis semanas. Cada grupo foi subdividido em dois subgrupos que receberam DXR (1mg/Kg) ou solução salina intraperitoneal na terceira e sexta semanas de tratamento. Os rins e fígado foram utilizados para isolamento do DNA, determinação da concentração de SAM, SAH e GSH, e análise da instabilidade genômica. Todos os grupos apresentaram o mesmo estado de metilação da região promotora do gene TP53, determinado pelo método de análise de restrição combinada com bissulfito (COBRA). Este fato poderia ser explicado pelo índice de metilação (razão SAM/SAH) que permaneceu inalterado, possivelmente devido a uma adaptação do ciclo da Met que manteve a concentração de SAM. A depleção de GSH não ocorreu quando DXR foi associada a dieta suplementada com Met. Portanto, a suplementação com Met manteve a concentração de GSH em ratos tratados com DXR. A dieta suplementada com Met não induziu instabilidade genômica e não alterou o dano ao DNA induzido pela DXR. Em conclusão, DXR induz depleção de GSH que é inibida pela suplementação com Met. Entretanto, a mesma suplementação não previne a instabilidade genômica induzida pela DXR. A dieta suplementada com Met aumenta a concentração de SAH renal sem alterar a concentração de SAM e GSH. Tanto a dieta suplementada quanto a DXR não induzem hipermetilação na região promotora do gene TP53. / The DNA methylation status is susceptible to changes when organisms are exposed to environmental agents such as food components and drugs. A methionine-rich (Met) diet may modulate S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations, which could change the DNA methylation status in the promoter region of tumor suppressor genes. Global hipomethylation and gene-specific hipermethylation are involved in genomic instability and it could result in DNA damage. This study intends to evaluate if a Met-rich diet associated with doxorubicin (DXR), an antitumoral drug that induces reactive species, result in changes in the methylation status of the TP53 gene promoter, in the SAM/SAH ratio, in glutathione levels (GSH) and in DNA damage. Forty male Wistar rats were separated into two groups: Met-rich diet (standard chow plus 2% Met), and control diet (standard chow) for six weeks. Each group was subdivided into another two groups that received DXR (1mg/kg) or saline intraperitoneally in the third and sixth weeks of the experiment. The kidneys and the liver were removed for DNA isolation, SAM, SAH and GSH determination, and genomic instability assay. All groups showed the same unmethylated status in the TP53 promoter according to the Combined Bisulfite Restriction Analysis (COBRA). This could be explained by the fact that the methylation index (SAM/SAH ratio) remained unchanged, possibly because of an adaptive Met pathway that maintains SAM levels. GSH depletion did not occur when DXR was associated with the Met-rich diet. As a matter of fact, the Met-rich diet improved GSH concentration in DXR-treated rats. Met-rich diet did not induce genomic instability, and it did not alter DNA damage induced by DXR. In conclusion, DXR induces GSH depletion which is inhibited by Met supplementation. However, Met-rich diet may not prevent genomic instability induced by DXR. A Met-rich diet increases SAH levels; however, it does not change GSH and SAM levels. Neither Met supplementation nor DXR induced DNA hypermethylation in the TP53 gene promoter.

Em julho de 2008, a ANVISA publicou um informe técnico esclarecendo um item importante da RE nº 1 (2005), que trata sobre os estudos de estabilidade de medicamentos. Este documento originou uma nova RDC de nº 58, publicada em dezembro de 2013, a qual estabelece limites para produtos de degradação em medicamentos. O objetivo do presente trabalho foi avaliar o comportamento do antineoplástico cloridrato de doxorrubicina frente a condições de decomposição (hidrólise ácida, básica, oxidação, temperatura e fotólise), a fim de se determinar suas principais vias de degradação e também elucidar as estruturas de seus principais produtos de degradação. Para isso foi desenvolvido e validado um método indicativo de estabilidade por HPLC-DAD-MS, o qual utiliza como fase estacionária uma coluna Luna C18(2) (150 mm x 3,0 mm, µm) com gradiente de fase móvel de tampão formiato de amônio 5 mmoles.L-1 pH 3 e metanol e fluxo de 0,3 mL.min-1. Ao longo do estudo foram encontrados diversos produtos de degradação, dentre eles a 7- deoxidehidrodoxorrubicinona, originada por hidrólise ácida e também o produto da degradação térmica de m/z 530, o qual foi encontrado nas análises do medicamento após expirado seu prazo de validade. Além disso, a avaliação da toxicidade in vitro de amostras contendo produtos de degradação de origem térmica indicou atividade citotóxica para células mononucleares. / ANVISA has published in July 2008, a technical sheet expaining an important item of the RE No. 1 (2005), which describes drugs stability studies. This document originated a new RDC No. 58, published in December 2013, which sets thresholds for degradation products in pharmaceuticals. The aim of this study was to evaluate the behavior for the antineoplastic doxorubicin when exposed to stress conditions (acid and base hydrolysis, oxidaton, photolysis and temperature) in order to determine the major pathaways of degradation and also to elucidate the stuctures of their main degradation products. To this was developed and validated a target stability indicatinf method by HPLC-DAD-MS, with Luna C18 (2) (150 mm x 3.0 mm. 3 µm) column as stationary phase and a mobile phase gradient composed of ammonium formate buffer 5 mmoles.L-1 and pH 3 and metanol with flow of 0.3 ml min-1. Throughout the study many degradation products was discovered, among them 7- deoxydehydrodoxorubicinone, formed by acid hydrolysis and also the main product of termal decomposition of m/z 530, wich was found in the analyzes of the medicine after the expiry of its validity were found. Furthermore, evaluation of the in vitro toxicity of samples containing degradation products of thermal decomposition was found to be citotoxic for mononuclear cells.

O carcinoma hepatocelular (CHC) é a neoplasia maligna primária mais comum do fígado, sendo a quinta mais frequente e a terceira causa de morte por câncer no mundo. Atualmente, nenhum protocolo com resultados satisfatórios no tratamento de CHC foi preconizado. Neste estudo foi determinado o potencial regenerativo e imunomodulador das células-tronco mesenquimais (CTM) associadas ou não aos quimioterápicos doxorrubicina e gencitabina, no modelo ortotópico de CHC em camundongos C57BL/6J. Foi realizado in vitro a determinação da atividade citotóxica dos quimioterápicos doxorrubicina e gencitabina em células de CHC murino (Hepa1c1c7), quantificação da peroxidação de lipídeos da membrana celular (TBARS) e análise das fases do ciclo celular e a expressão de marcadores por citometria de fluxo. A IC50% em células de carcinoma hepatocelular murino (Hepa1c1c7) foi de 1,85 uM para doxorrubicina e 20,8 ?M para gencitabina. A quantificação de TBARS na linhagem celular de CHC murino (Hepa1c1c7) tratados com os quimioterápicos doxorrubicina e gencitabina demonstrou efeito deletério apenas quando tratados nas concentrações (55 a 250 uM) com a doxorrubicina e (1,75 a 7 uM) com gencitabina. Após tratamento com os quimioterápicos, ocorreram modificações nas populações celulares, com o aumento da fase sub-G1 e G0/G1 e diminuição fases S e G2/M. As CTM, apresentaram-se aderentes aos frascos de cultura com morfologia semelhante a fibroblastos e expressão positiva para marcadores CD44, CD73, CD90 e CD105 e negativa para células-tronco hematopoiéticas da medula óssea: CD11b, CD45 e CD117. O modelo experimental tumoral ortotópico de CHC foi estabelecido no 21° dia após a inoculação das células tumorais. Os efeitos da terapêutica com a CTM mostraram aspectos significativos na redução da massa tumoral (30,5%), os demais grupos experimentais tratados com quimioterapia sistêmica associada à terapia celular também evidenciaram resultados significativos na redução da massa tumoral e atenuação dos efeitos de toxicidade sistêmica. As células tumorais extraídas dos tumores hepáticos encontram-se preferencialmente na fase sub-G1 nos grupos de animais tratados com CTM em associação aos quimioterápicos gencitabina (29%) e doxorrubicina (21%), enquanto o tratamento isolado com a doxorrubicina foi de 22% e de 15% para a gencitabina, além de induzir significativamente diminuição das fases S e G2/M. No grupo de animais tratado com CTM em associação ao quimioterápico gencitabina há um aumento da expressão das caspases 3 e 8, e dos marcadores CD44 e IL-1R. O grupo de animais tratados com CTM apresentou aumento na expressão dos marcadores CD90 e CD14. Os resultados obtidos no tratamento do modelo ortotópico de CHC com CTM associadas aos quimioterápicos doxorrubicina e gencitabina apresentaram eficácia na redução da massa tumoral e atenuação dos efeitos colaterais causados pelo tratamento quimioterápico sistêmico, além do seu potencial efeito imunomodulador / Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, the most frequent fifth and the third leading cause of cancer death in the world. Currently, no protocol with satisfactory results in the treatment of CHC was recommended. In this study we determined the regenerative potential immunomodulatory and mesenchymal stem cells (MSCs) associated or not to chemotherapy doxorubicin and gemcitabine, in the orthotopic model of CHC in C57BL / 6J mice. Was determined in vitro the cytotoxic activity of doxorubicin and gemcitabine chemotherapy in a murine cells (Hepa1c1c7), quantification of cell membrane lipid peroxidation (TBARS) and analysis of the phases of the cell cycle and expression of markers by flow cytometry. IC50% in murine carcinoma cells (Hepa1c1c7) was 1.85 uM and 20.8 uM for doxorubicin to gemcitabine. The quantification of TBARS in cell line (Hepa1c1c7) treated with the chemotherapeutic doxorubicin and gemcitabine showed a deleterious effect only when treated at concentrations (55-250 uM) doxorubicin and (1.75 to 7 uM) with gemcitabine. After treatment with chemotherapeutic agents, there were changes in the cell population, with increased phase sub-G1 and G0 / G1 phase and reduced S and G2 / M. MSC presented to adhere to culture flasks with morphology similar to fibroblasts and positive expression of markes: CD44, CD73, CD90 and CD105 and negative hematopoietic stem cells from bone marrow: CD11b, CD45 and CD117. The tumor orthotopic experimental model of HCC was established on day 21 after tumor cell inoculation. The effects of therapy CTM showed significant aspects in reducing the tumor mass (30.5%), the other experimental groups treated with systemic chemotherapy associated with cell therapy also showed significant results in reducing the tumor mass and mitigation of systemic toxicity effects. The extracted tumor cells of liver tumors are preferably in the sub-G1 phase in groups of animals treated with MSC in association with gemcitabine (29%) and doxorubicin (21%) chemotherapy, whereas the single treatment with was 22% doxorubicin and 15% for gemcitabine in addition to significantly induce reduction of phases S and G2 / M. In the group of animals treated with MTC in combination with gemcitabine chemotherapy there is an increased expression of caspase 3 and 8 and IL-1R and CD44. The group of animals treated with MSCs showed increased markers expression of CD90 and CD14. The results obtained in the treatment of orthotopic HCC model with MSC associated with the chemotherapeutic doxorubicin and gemcitabine showed efficacy in reducing tumor burden and attenuation of the side effects caused by systemic chemotherapy, as well its potential immunomodulatory effect