This study aims to prepare theranostic nanoparticles (NPs) that are expected to increase cancer diagnostics and therapeutic efficacy. We prepared the NPs constituting carbon dots (CDs) as an imaging agent, folic acid as a targeting agent, doxorubicin (DOX), or gemcitabine (GEM) as chemotherapy agents. The NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. Through ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform-infrared spectroscopy, the fabrication of these NPs was confirmed. It was discovered that the high drug loading efficiency is the noncovalent series while the high drug loading capacity is the covalent series The in-vitro pH-dependent drug release data indicate the NPs release more drugs at around pH 5.0 than at pH 7.4. The NPs sizes are between 2-5 nm. The Cell viability was investigated using the Alamar Blue assay and the three NPs complexes exhibited strong therapeutic efficacy against MDA-MB-468 breast cancer cells as compared with CDs-drug.

No description available.

No description available.

The main aim of this study is to synthesize theranostic nanoparticles (NPs) that will drastically increase the diagnostics and therapeutic efficacy for cancer. In this research, we had prepared the NPs which constitute carbon dots (CDs), the imaging agent, Folic acid, the targeting agent, and Doxorubicin (DOX) or Gemcitabine (GEM) as the chemotherapy agents. The prepared NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. The spectroscopy, ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy, and Fourier transform-infrared spectroscopy (FT-IR), were used to confirm the successful fabrication of these complexes. Through UV-vis analysis, the drug loading capacity (DLC) and drug loading efficiency (DLE) of the complexes were determined. The noncovalent series had a higher DLE of about 83% while the covalent series showed higher DLC, 70% on average indicating high drug content. The in-vitro pH-dependent drug release shows that the noncovalent FA-CDs-DOX and the covalent FA-CDs-GEM series release more drugs into the cancer cells (pH of 5.0) than into healthy normal (pH of 7.4). The sizes of NPs were measure around 2-5 nm with Dynamic light Scattering (DLS). The toxicity of CDs, CDs-drug, and FA-CDs-drug on MDA-MB468 breast cancer cell was tested through the methylthiazolytetrazolium (MTT) assay and found that the FA bonded NPs exhibited strong therapeutic efficacy. More pharmaceutical data towards the cancer cells are investigated by our research collaborators – the pharmaceutical department at ETSU and Xavier University at Louisiana.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies because it is often diagnosed at a late disease stage and has a poor response rate to currently available treatments. Therefore, it is critical to develop new therapeutic approaches that will enhance the efficacy and reduce the toxicity of currently used therapies. Here we aimed to evaluate the therapeutic potential and mechanisms of action for piperlongumine (PL), an alkaloid from long pepper, in PDAC models. We postulated that PL causes PDAC cell death through oxidative stress and complements the therapeutic efficacy of chemotherapeutic agents in PDAC cells. First, we determined that PL is one of the most abundant alkaloids with antitumor properties in the long pepper plant. We also showed PL in combination with gemcitabine, a chemotherapy agent used to treat advanced pancreatic cancer, reduced tumor weight and volume compared to vehicle-control and individual treatments. Further, biochemical analysis, including RNA sequencing and immunohistochemistry, suggested that the antitumor activity of PL was associated with decreased cell proliferation, induction of cell cycle arrest, and oxidative stress-induced cell death. Moreover, we identified that c-Jun N-terminal kinase (JNK) inhibition blocks PL-induced cell death, translocation of Nrf2, and transcriptional activation of HMOX1 in PDAC. Finally, high-throughput drug and CRISPR screenings identified potential targets that could be used in combination with PL to treat PDAC cells. Collectively, our data suggests that cell cycle regulators in combination with PL might be an effective approach to combat pancreatic cancer. / NIH

No description available.

20 svenska barn diagnostiseras årligen med barntumören neuroblastom (NB). Läkemedelsresistens och intratumoral heterogenitet försvårar behandlingen och 50-60% av hög-risk NB-patienter drabbas av återfall. Subpopulationer av resistenta celler har identifierats i hög-risk NB-cellinjer, vilket belyser behovet av nya behandlingsalternativ. Dessa celler har visats vara känsliga mot antimetaboliter, som är S-fas specifika läkemedel. Syftet med detta projekt är att utvärdera antimetaboliter som singelbehan-dling eller i kombination med kemoterapi som en ny behandlingsmetod i resistenta NB-celler. Cellvia-bilitiet, cellåterväxt efter långtidsbehandling och cellcykelarrest samt cellcykelns förlopp analyserades på en panel bestående av de fem TP53-muterade ultra-högrisk NB-cellinjerna Kelly, SK-N-DZ, SK-N-AS, BE(2)-C och SK-N-FI. Läkemedlena som utvärderades var kemoterapierna doxorubicin (doxo) och cisplatin samt antimetaboliterna cytarabine (Ara-C), gemcitabine, 5-fluorouracil (5-FU) och hy-droxyurea (HU). Resultaten indikerar att Kelly och SK-N-FI var de cellinjer som var mest känsliga mot kombinationer av doxo tillsammans med antimetaboliter. Detta styrktes ytterligare av analysen av cellcykelarrest som visade att dessa cellinjer föredrar att arrestera i S-fas. Cellinjerna SK-N-DZ, SK-N-AS och BE(2)-C var mindre känsliga mot kombinationsbehandlingarna och visade preferens för att arrestera i andra faser av cellcykeln. Eftersom cellinjerna visade variationer i cellcykelarrest så föreslår vi en utvärdering av trippelkombinationer med läkemedel som är G2/M-fas specifika för att ytterligare eliminera resistenta subpopulationer. / Every year, 20 Swedish children are diagnosed with the pediatric tumor neuroblastoma (NB). Therapy resistance and intratumor heterogeneities complicate treatment of NB and ultimately, 50-60% of high- risk NB patients relapse. Subpopulations of resistant cells have been identified in high-risk NB cell lines, which elucidates the need for novel treatment options. These cells have been suggested to be sensitive to the S-phase specific drugs antimetabolites. The objective of this project is to evaluate antimetabolites as monotherapy and in combination with chemotherapy as a novel treatment option in high-risk NB. Analyses of cell viability, cell regrowth following long-term treatment, and cell cycle progression and mitotic arrest were performed in a panel of five TP53 mutated ultra-high risk NB cell lines, Kelly, SK- N-DZ, SK-N-AS, BE(2)-C, and SK-N-FI. The evaluated drugs were the chemotherapies doxorubicin (doxo) and cisplatin, and the antimetabolites cytarabine (Ara-C), gemcitabine, 5-fluorouracil (5-FU), and hydroxyurea (HU). Obtained results indicated that out of the tested cell lines, Kelly and SK-N-FI are the most sensitive to combinations of doxo with antimetabolites. This was further corroborated via analysis of cell cycle progression and mitotic arrest which demonstrated that Kelly and SK-N-FI have a preference for S-phase arrest. Cell lines SK-N-DZ, SK-N-AS, and BE(2)-C were less sensitive to combination treatments and showed preference for arrest in other phases of the cell cycle. Since resistant cell lines show variations in mitotic arrest, we suggest evaluating triple combinations with targeted treatments for G2/M-phase, in order to further eliminate resistant subpopulations.

L’adénocarcinome pancréatique (AP) connaît une forte augmentation d’incidence, qui en fait la quatrième cause de mortalité par cancer avec un pronostic extrêmement sombre, moins de 5% des patients étant en vie à 5 ans. De nombreuses avancées dans la compréhension de l’oncogénèse pancréatique notamment sur les aspects génétiques, immunitaires et sur les interactions cellulaires du stroma tumoral ont permis d’envisager le développement de nouvelles stratégies de traitement. Cependant malgré des résultats pré-cliniques très encourageants aucune de ces stratégies n’a encore permis l’émergence d’un traitement plus efficace que la chimiothérapie standard. Ce travail de thèse a abordé 2 approches thérapeutiques innovantes distinctes mais potentiellement complémentaires dans le traitement de l’adénocarcinome pancréatique en étudiant in vitro (cultures cellulaire 2D et 3D) et in vivo (modèles ectopiques et orthotopiques) les effets sur la croissance tumorale de l’inhibition d’un acteur important du stress oxydant (la voie Nrf2) d’une part, de la combinaison d’une chimiothérapie liposomale et d’un agent physique, la cavitation ultrasonore, d’autre part. La cavitation ultrasonore est un effet mécanique des ultrasons permettant d’augmenter l’internalisation de molécules ou de gènes dans les cellules. Dans cette thèse, la faisabilité et l'efficacité de la combinaison d’une chimiothérapie liposomale ciblée par la cavitation ultrasonore a été évaluée dans des modèles murins orthotopiques d’AP. Un système de délivrance d’ultrasons a été adapté afin d’appliquer une cavitation inertielle focalisée sur des xénogreffes d’AP créées après l'injection de doxorubicine liposomale (L-DOX) selon une étude pharmacocinétique préliminaire réalisée dans un modèle murin. La L-DOX, conçue à base de phospholipides non saturés de dioleoylphosphatidyléthanolamine, connue pour être stable dans la circulation sanguine, a été choisie afin de maximiser son accumulation et le relargage du principe actif lors de la délivrance des ultrasons. Nous montrons que l’association de la L-DOX à la cavitation inertielle permet de réduire in vivo le volume tumoral dans un modèle orthotopique d’AP chez la souris nude. La cavitation inertielle peut donc augmenter l'effet antitumoral des liposomes porteurs de chimiothérapie avec un effet mécanique minimal sur le tissu environnant la tumeur.Des études récentes suggèrent que Nrf2 est une cible de choix pour vaincre la chimiorésistance de l’AP. Des méthodes in vitro et in vivo ont été utilisées afin d’examiner l'effet du brusatol associé à des agents chimiothérapeutiques de référence sur la mort cellulaire et son impact sur le stress oxydant. Nous montrons que l’inhibition de la voie Nrf2 par le brusatol, un composé naturel issu de Fructus Bruceae, potentialise les effets de la chimiothérapie et permet l’inhibition de la croissance tumorale in vitro sur des lignées cellulaires d’AP cultivées en 2D et 3D. Cette inhibition s’accompagne d’une modulation du stress oxydant dont témoignent l’augmentation des espèces réactives de l’oxygène (ROS) et la diminution du glutathion (GSH). In vivo, la combinaison du brusatol et de l'oxaliplatine a réduit le volume tumoral dans deux modèles murins de xénogreffe d’AP. Ces résultats suggèrent l'efficacité du brusatol pour lutter contre la chimiorésistance et renforce l’hypothèse d’un rôle clinique potentiel de l’inhibition de Nrf2 comme adjuvant à la chimiothérapie dans l’AP. Un travail clinique a également été mené en parallèle sur une modalité de traitement physique innovante, la radiofréquence endobiliaire, dans la prise en charge endoscopique de l’ampullome vatérien, tumeur rare à la croisée entre le tube digestif et le système biliopancréatique. Les résultats de cette étude prospective multicentrique seront également présentés dans cette thèse. / Pancreatic ductal adenocarcinoma (PDAC) has increased in incidence over the past decade, leading it to be the fourth lethal cause of cancer in the world with a very poor prognosis, since less than 5% of patients are alive at 5 years. Many advances in the understanding of pancreatic tumorigenesis, notably on the genetic, immune and cellular stroma interactions of the tumor, have led to the development of new treatment strategies in the last decade. However, despite very encouraging pre-clinical results, none of these strategies has yet led to the emergence of a truly effective treatment in comparison with standard chemotherapy. This thesis focused on two innovative therapeutic modalities in the treatment of PDAC at a preclinical stage by studying in vitro (2D and 3D cell cultures) and in vivo (ectopic, orthotopic xenografts) the effects on the tumor growth of an inhibitor of the Nrf2 pathway (involved in oxidative stress), on the first hand, and of a physical element, ultrasound cavitation associated with liposomal chemotherapy, on the second hand. Ultrasound cavitation is a mechanical effect of ultrasound to increase the uptake of molecules or genes in cells. The feasibility and effectiveness of the combination of liposomal chemotherapy targeted by ultrasonic cavitation was evaluated in murin orthotopic models of PDAC. An ultrasound delivery system has been adapted to apply focused inertial cavitation to PDAC xenografts created after the injection of liposomal doxorubicin (L-DOX) according to a preliminary pharmacokinetic study carried out in the murine model. L-DOX, designed on unsaturated phospholipids of dioleoylphosphatidylethanolamine, was known to be stable in the bloodstream and to maximize its accumulation and release of the active drug during ultrasound delivery. This thesis shows that this therapeutic combination (L DOX and inertial cavitation) makes it possible to reduce the tumor volume in vivo in a nude mouse orthotopic model of PDAC. Inertial cavitation may be generated to increase the therapeutic effect of chemotherapybearing liposomes accumulated in the tumor with minimal mechanical effect on the surrounding tissue. Recent studies strongly suggest that Nrf2 is an ideal target against chemoresistance of PDAC. In vitro and in vivo methods were combined to examine the effect of brusatol associated with chemotherapeutic agents on cell death in addition to its impact on oxidative stress (reactive oxygen species and gluthation levels). This thesis demonstrates that the inhibition of the Nrf2 pathway via brusatol, a natural compound derived from Fructus Bruceae, potentiates the effects of chemotherapy and allows the inhibition of tumor growth in vitro on PDAC cell lines. This inhibition is accompanied by a modulation of oxidative stress by brusatol, with increasing ROS and decreasing GSH. In vivo, the combination of brusatol and oxaliplatin reduced tumor volume in two mouse models of PDAC xenograft. These results suggest the efficacy of using brusatol to combat chemoresistance and reinforce the idea that brusatol could be developed as an adjuvant to chemotherapy in PA. Clinical work was also carried out in parallel on an innovative physical treatment modality, endobiliary radiofrequency, in the management of adenoma of the ampoule of Vater, a rare tumor located between the digestive and the bilio-pancreatic systems. The results of this work will also be presented in this thesis.

Successful treatment of cancer with adoptive immunotherapy (AIT) is dependent on the ability to produce large numbers of tumor-specific, functional T cells. The purpose of this thesis is to explore ways in which T cell expansion could be augmented. We have focused on exploring alternate gamma chain cytokines as stimulators of T cell proliferation and differentiation in addition to investigating the potential usefulness of gemcitabine (GEM) in abrogating the immunosuppressive effects of myeloid derived suppressor cells (MDSCs). B16 melanoma sensitized draining lymph node cells that have been activated in vitro with bryostatin-1 and ionomycin (B/I) were expanded in either IL-7/15 or in IL-2. We found that IL-7/15 was superior to IL-2 in expanding T cells for AIT of pulmonary metastases. Expansion of antitumor T cells was also improved by suppressing accumulation of MDSCs in mice bearing 4T1 mammary carcinoma using GEM. GEM directly inhibits both 4T1 mammary carcinoma cells and MDSCs. Its inhibition of MDSCs rescued tolerant T cells, augmenting both expansion and response to tumor antigen.

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