Le myélome multiple (MM) est une hémopathie dont la croissance et la prolifération sont liés à une variété de facteurs de croissance, y compris « insulin-like growth factor type 1 » (IGF-1). Bortézomib est le premier inhibiteur protéasome ayant une activité anti-tumorale significative dans le myélome multiple. Nous avons analysé l'impact de l'IGF-1 recombinant associé à l'inhibiteur du protéasome bortezomib sur des lignées humaines de MM, in vivo et sur des cellules de myélome frais humaines ex vivo. Nous avons montré que l'IGF-1 améliore l'activité cytotoxique du bortezomib in vitro, in vivo et ex vivo. Nous avons montré que l'accroissement de la toxicité peut être inhibé par la présence d'un anticorps monoclonal dirigé contre le récepteur de l'IGF-1 (IGF1-R). IGF-1 renforce l'activité cytotoxique des autres inhibiteurs de protéasome, y compris MG115, MG132, PSI et epoxomicin. Nos résultats confirment le fait que l'IGF-1sensibilise des cellules de myélome à l'activité cytotoxique des inhibiteurs du protéasome tels que le bortezomib, en raison du niveau accru du stress de réticulum endoplasmique et l'induction de la une réponse protéine dépliée (UPR) / Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib in MM cell lines, in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro, in vivo and ex vivo. We showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 enhances the cytotoxic activity of other proteasome inhibitors, including MG115, MG132, PSI and epoxomicin. Our results support the fact that IGF-1sensitize myeloma cells to the cytotoxic activity of proteasome inhibitors such as bortezomib, as a consequence of enhanced level of endoplasmic reticulum stress and the induction of an unfolded protein response (UPR)

Mieloma Múltiplo é a segunda doença com maior prevalência nas doenças malignidades hematológica, incurável com média de sobrevivência de 3-5 anos. MM é uma malignidade das células do plasma caracterizada pela destruição e reabsorção óssea e supressão da formação do osso. A quimiocina SDF-1 (CXCL12) e seu receptor CXCR4 têm um importante papel direcional na migração, homing das células do plasma em mieloma múltiplo e mobilização das células de MM para fora da medula óssea. A talidomida tem sido usada com êxito no tratamento de pacientes com mieloma múltiplo. Neste estudo verificamos o efeito da talidomida na expressão da quimiocina SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo e em linhagem de células de mieloma múltiplo humano (RPMI-8226) tratados e sem tratamento de talidomida. Nossos resultamos mostraram uma expressão heterogênea na expressão da quimiocina SDF-1 e seu receptor CXCR4 nos pacientes com mieloma múltiplo estudado (n= 79). Entretanto, pacientes com mieloma múltiplo tratados com talidomida mostraram uma baixa expressão da quimiocina SDF-1 e seu receptor CXC4 quando comparados com pacientes recém diagnosticados para mieloma múltiplo e pacientes com mieloma múltiplo tratados com outros medicamentos. Nossos resultados sugerem que o tratamento com talidomida induz uma baixa regulação na expressão no ligante SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo / Multiple Myeloma (MM) is a second most prevalent hematological malignancy and remains incurable with a median survival of 3-5 years. MM is a plasma cell malignancy characterized by devastating bone destruction due to the enhanced bone resorption and suppressed bone formation. The chemokine stromal-derived factor-1 (SDF-1) and its receptor CXCR4 play an important role in directional migration, homing of plasma cells in multiple myeloma (MM) and mobilization of MM cells out of the bone marrow. The drug thalidomide has been successfully used in the treatment of patients with MM. In this study, we assessed the effect of thalidomide on SDF-1 and CXCR4 expression in MM patients and human myeloma-derived cell line, RPMI 8226 treated with or without thalidomide. A heterogeneous expression pattern of chemokines SDF-1 and CXCR4 receptor were observed for all MM patients studied. However, patients treated with thalidomide showed a significantly decrease in expression of SDF-1 and CXCR4 as compared to newly diagnosed MM patients and MM patients treated with other drugs. RPMI 8226 cell line treated with 10, 20 and 100µM thalidomide also demonstrated decrease in SDF-1 and CXCR4 expression as compared with cell control (RPMI-8226 without thalidomide). Ours results indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1 in multiple myeloma

Das Knochenmarkmikromilieu produziert eine Reihe von unterschiedlichen Wachstumsfaktoren, die für das maligne Wachstum und die Medikamentenresistenz von Myelomzellen von grosser Bedeutung sind. Einer der wichtigsten Faktoren, der in manchen experimentellen Systemen sogar als essentiell für das Wachstum und Überleben von Myelomzellen beschrieben wurde, ist Interleukin-6. Aus diesem Grund könnte die Entwicklung von Substanzen, die die Wirkung von IL-6 oder dem IL-6 Rezeptor inhibieren von Bedeutung für die Therapie des Myeloms sein. In dieser Arbeit wurde die Wirkung des IL-6 Rezeptorantagonisten SANT7 auf das Überleben der IL-6 abhängigen Myelomzellinie INA-6 sowie primären Myelomzellen in Gegenwart oder Abwesenheit von primären humanen Knochenmarkstromazellen (KMSZ) untersucht. Von besonderem Interesse war hierbei die Frage ob SANT7 die wachstumsinhibitorische Wirkung von Dexamethson (Dex) und All-Trans-Retinolsäure (ATRA) verstärken kann. Keine der drei Substanzen, SANT7 eingeschlossen, konnte bei alleiniger Applikation in Gegenwart von primären humanen KMSZ eine nennenswerte Wachstumsinhibition induzieren. Wenn jedoch Dex und ATRA mit SANT7 kombiniert wurden konnte sowohl in INA-6 als auch primären Myelomzellen eine starke Wachstumsinhibition erzielt werden. Dieser Effekt beruht sowohl auf Apoptose als auch eines Zellzyklusarrests. / The bone marrow microenvironment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is interleukin-6 (IL-6). Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have a potential therapeutic value for the treatment of multiple myeloma. In this work the effect of the IL-6 receptor antagonist SANT7 on growth and survival of the IL-6 dependent MM cell lines INA-6 as well as primary MM cells in the presence or absence of bone marrow stromal cells (BMSC) was analyzed. Of particular interest was the question whether SANT7 might enhance the growth inhibitory effects of dexamethasone (Dex) and all-trans retinoic acid (ATRA). None of the drugs, when tested as a single substance, including SANT7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, if Dex and ATRA were given in combination with SANT7 a strong growth inhibition was achieved in INA-6 and primary MM cells. This effect is due to cell cycle arrest and induction of apoptosis.

Das Multiple Myelom (MM) ist eine hämatologische Erkrankung, welche sich durch eine Akkumulation von malignen Plasmazellen im Knochenmark auszeichnet und eine gestörte Hämatopoiese und Osteolyse zur Folge hat. Komplexe molekulare Interaktionen zwischen MM-Zellen und der Mikroumgebung/Nische im Knochenmark (bone marrow microenvironment, BMM) führen zu einer Aktivierung von verschiedenen Wachstums-, Überlebens- und anti-apoptotischen Signalwegen, die zur Entstehung bzw. Wirkstoffresistenz von MM-Zellen beitragen. IL-6R/STAT3, Ras/MAPK und PI3K/Akt sind die drei wichtigsten Signalwege, die mit dem Wachstum und der Entwicklung des MM assoziiert sind. Auf der anderen Seite sind Myelomzellen insensitiv gegenüber einer Blockade des IL6R/STAT3-Signalweges bzw. des Ras/MAPK-Signalwegs in der Gegenwart von Knochenmarksstromazellen (bone marrow stroma cells, BMSCs), was die Entbehrlichkeit dieser beiden Signalwege unter Ko-Kultur-Bedingungen nahelegt. Interessanterweise aber induziert die gleichzeitige Unterbrechung der IL6R/STAT3 und Ras/MAPK Signalwege Apoptose in MM-Zellen. Ziel der Arbeit war die Identifizierung und Analyse von Zielgenen, die von beiden Signalwegen und nicht durch einen Signalweg alleine reguliert werden. Genexpressionsanalysen zeigten eine deutliche Herunterregulierung der Proteine Hsp90alpha und Hsp90beta nach einer gleichzeitigen Inhibition der IL6R/STAT3 und Ras/MAPK Signalwege. In Hinblick auf die zentrale Rolle von Hsp90 in der Tumorbiologie fokussiert sich die vorliegende Arbeit auf die Erforschung der Rolle von Hsp90 im Multiplen Myelom. Die siRNA-vermittelte Herunterregulation der Proteinexpression von Hsp90-Proteinen zeigte, daß das Ausschalten von HsP90alpha alleine nur zu einer moderaten Apoptoseinduktion in INA-6- und MM.1s-Zellen führte. Die gleichzeitige Herunterregulation von HsP90beta hingegen führte zu einer Verstärkung dieses Effektes und deutet darauf hin, daß beide Proteine miteinander kooperieren. Die pharmakologische Inhibition der Hsp90-Funktion mittels eines neuen Hsp90-Inhibitors (17-DMAG) führte zu einer Verringerung von phospho-ERK1/2, zur Degradation von STAT3 und zu einem verminderten Überleben von MM-Zellen. Die pro-apoptotischen Effekte der gestörten Hsp90-Funktion konnten weder durch BMSCs und Osteoklasten noch durch ECs (??) abgeschwächt werden, obwohl für ECs beschrieben wurde, daß sie zum Wachstum und Überleben von MM-Zellen beitragen können. Diese Beobachtungen deuten auf einen positiven Rückkopplungskreislauf zwischen HsP90alpha/beta und den wichtigsten Signalwegen hin, welcher das Überleben von MM-Zellen gewährleistet. Desweiteren zeigten immunhistologische Analysen, daß Hsp90-Proteine im Vergleich zu MGUS (??) bzw. normalen Plasmazellen in MM-Plasmazellen hochreguliert sind. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit die essentielle Rolle von Hsp90-Proteinen für die Überlebensfähigkeit von MM-Zellen. Ein neuer Mechanismus der Hsp90-Regulation durch das Zusammenwirken der Signalwege IL6R/STAT3 und Ras/MAPK in MM-Zellen konnte gezeigt werden. Darüber hinaus deuten die Ergebnisse darauf hin, daß ein positiver Rückkopplungskreislauf zwischen Hsp90-Proteinen und den wichtigsten Signalwegen existiert, welcher zum Wachstum und zur Entwicklung von MM-Zellen beiträgt. Die Inhibition der Hsp90-Funktion durch den pharmakologischen Inhibitor 17-DMAG führte zum Absterben von MM-Zellen und der pro-apoptotische Effekt der Hsp90-Depletion konnte nicht durch unterstützende BMM-Zellen aufgehoben werden. Diese Beobachtungen untermauern die multifunktionelle Rolle von Hsp90 in der MM-Biologie und zeigen die Wichtigkeit der Entwicklung neuer therapeutischer Wirkstoffe zur Inhibition der Hsp90-Funktion bei der Behandlung des MM. / Multiple myeloma (MM) is a haematological malignancy characterised by the accumulation of malignant plasma cells in the bone marrow leading to impaired haematopoiesis and osteolytic bone destruction. Intricate molecular interactions between MM cells and the BMM activate a diverse set of growth, survival and anti-apoptotic signaling cascades that mediate tumor progression and drug resistance. IL-6R/STAT3, Ras/MAPK and PI3K/Akt are the three major signal transduction pathways that are associated with MM growth and progression. However, myeloma cells have shown independence from IL-6R/STAT3 blockade or insensitivity towards Ras/MAPK pathway inhibition in the presence of BMSCs, indicating the dispensability of both in co-culture conditions. Interestingly, concomitant disruption of both IL-6R/STAT3 and Ras/MAPK pathways was successful to drive MM cells into significant apoptosis. This study aimed to identify and analyse the downstream target genes that are regulated by both pathways and not by either pathway alone. Gene expression profiling revealed prominent downregulation of Hsp90alpha and Hsp90beta proteins after combined inhibition of the IL-6R/STAT3 and Ras/MAPK pathways. Owing to the important role played by Hsp90 in cancer biology, this study was narrowed down to investigate the role of Hsp90 in MM. Specific siRNA-mediated knockdown of Hsp90 proteins showed that although knockdown of Hsp90beta was sufficient to induce moderate apoptosis in INA-6 and MM.1s cells, the effect was more pronounced when both Hsp90 proteins were targeted, indicating co-operation between them. Pharmacological inhibition of Hsp90 function by using a novel Hsp90 inhibitor (17-DMAG) down-regulated the levels of pERK1/2 and led to degradation of STAT3 and decreased viability of MM cells. The pro-apoptotic effects of compromised Hsp90 function could not be alleviated by either BMSCs, OCs or ECs, which are well-known to support myeloma growth and survival. These observations point to the existence of a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supporting MM cell survival. Furthermore, immunohistochemical analysis unveiled the up-regulated status of Hsp90 proteins in MM PCs as compared to MGUS or normal PCs. Taken together, the results of this study explain the critical contribution of Hsp90 proteins to MM cell survival. A novel mechanism of Hsp90 regulation by co-operation between the IL-6R/STAT3 and Ras/MAPK pathways was discovered in myeloma cells. There is also strong evidence of the existence of a positive feedback loop between Hsp90alpha/beta proteins and major signaling pathways supporting MM growth and progression. Inhibition of Hsp90 function by using the Hsp90 inhibitory drug 17-DMAG proved to be lethal for myeloma cells and the pro-apoptotic effects of Hsp90 blockade could not be reversed by the presence of cells from the supportive BMM. These observations highlight a multi-functional role of Hsp90 in MM biology and strongly strengthen the notion that therapeutic strategies targeting Hsp90 may open new perspectives for anti-myeloma drug development.

Die Hochdosischemotherapie stellt für Patienten unter 60 Jahren die Standardtherapie des multiplen Myeloms dar. Nach 600 Hochdosischemotherapiezyklen mit autologer peripherer Stammzelltransplantation zeigten sich erstmals Grundkrankheit, Alter und Zahl der transplantierten Stammzellen als unabhängige Einflußfaktoren für die Rate der dokumentierten Infektionen. Die Dauer der schweren Mukositis beeinflußte sowohl die Rate der Infektionen, als auch die therapieassoziierte Mortalität signifikant. Die Angiogenese im Knochenmark stellte bei Patienten mit multiplem Myelom einen unabhängigen Prognosefaktor für das Überleben dar. Myelom-Patienten wiesen im Vergleich zu gesunden Probanden eine signifikant erhöhte bFGF(basic fibroblast growth factor)-Konzentration im Serum auf. Es zeigte sich ein signifikanter Anstieg der bFGF Konzentration parallel zur Progression der Erkrankung vom Stadium I bis zum Stadium III. Eine effektive Chemotherapie ging mit signifikanten Reduktionen der Serum-Konzentrationen von drei angiogen wirksamen Zytokinen VEGF (vascular endothelial growth factor), bFGF und HGF (hepatocyte growth factor) sowie der Angiogenese im Knochenmark einher. Die Induktion der Knochenresorption stellt eines der wichtigsten weiteren Charakteristika von malignen Plasmazellen dar. In einer vergleichenden Untersuchung zeigte sich, daß unter den Knochenstoffwechselparametern ICTP (carboxyterminales quervernetztes Typ I Kollagen Telopeptid), NTx (aminoterminales Kollagen Typ I Telopeptid) und DPD (Desoxypyridinolin) das erstgenannte sich am besten zur Kontrolle der Progression eignet und dieser Parameter auch einen signifikanten Prognosefaktor für die Überlebenszeit darstellt. Die Hinweise, daß eine frühzeitige Bisphosphonattherapie die Progression des multiplen Myeloms hemmen könnte, hat uns veranlaßt, die erste prospektiv randomisierte Studie einer Bisphosphonattherapie beim multiplen Myelom im Stadium I in Zusammenarbeit mit der Deutschen Studiengruppe Multiples Myelom zu initiieren. Darüber hinaus wird in dieser Arbeit auf die prognostische Relevanz zytogenetischer und molekulargenetischer Befunde beim multiplen Myelom sowie auf die Möglichkeit immunphänotypischer Untersuchungen zur Differenzierung zwischen multiplem Myelom und monoklonaler Gammopathie unbestimmter Signifikanz (MGUS) eingegangen. / High-dose chemotherapy is the standard treatment for patients with multiple myeloma younger than 60 years. In 600 cycles of high-dose chemotherapy with autologous blood stem cell transplantation, we found that the type of the malignant disease, age and number of transplanted stem cells were independent prognostic factors for the rate of documented infections. The duration of severe mucositis was significantly associated both with the rate of infections and treatment-related mortality. The angiogenesis in bone marrow was found to be an independent prognostic factor for survival in patients with multiple myeloma. Myeloma patients had significantly elevated serum bFGF (basic fibroblast growth factor) levels in comparison to healthy controls. Serum bFGF levels increased parallel to the progression of multiple myeloma from stage I to stage III. Effective chemotherapy was associated with significant reductions in serum levels of three angiogenic cytokines VEGF (vascular endothelial growth factor), bFGF and HGF (hepatocyte growth factor) and the bone marrow angiogenesis. The induction of bone resorption is one of the most important characteristics of malignant plasma cells. In a comparative analysis of bone resorption parameters ICTP (carboxy-terminal telopeptide of type-I collagen), NTx (amino-terminal collagen type-I telopeptide) and DPD (deoxypyridinoline) we found that ICTP was the most suitable marker for the detection of progression in multiple myeloma and that ICTP was a significant prognostic factor for survival in this disease. The findings that an early start of a treatment with bisphosphonates might delay the progression of multiple myeloma has lead us to start the first prospective randomized trial of bisphosphonate treatment in stage I in collaboration with the German Study Group Multiple Myeloma. Furthermore the prognostic relevance of cytogenetic and moleculargenetic findings in multiple myeloma and the use of immunophenotypic analysis for the differentiation of multiple myeloma and monoclonal gammopathy of undetermined significance (MGUS) was discussed in this work.

Made available in DSpace on 2017-07-21T19:59:56Z (GMT). No. of bitstreams: 1 Paula Josiane Janowski.pdf: 1415528 bytes, checksum: b8cff156f647bfc75021877c6b016d7b (MD5) Previous issue date: 2012-03-13 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The multiple myeloma (MM) reaches plasma cells that are completely different from each other, that produce high amounts of immunoglobulins. The genetic changes are multiple and complexes, affecting the disease course and the sensibility of the tumorous cells to the treatment. The bone marrow constitutes the ideal microenvironment for the disease development, secreting growth factors that are indispensable for the proliferation, survival and migration of tumourous cells. This project is evaluating the pravastatin effect, drug that inhibits the hydroxymethylglutaryl coenzime reductase (HMG-CoA reductase), in MM cells, from RPMI 8226 line. The cells were kept in RPMI culture media, added by 10% of fetal bovine serum in a temperature of 37ºC in atmosphere of 5% of CO2. After the addiction of pravastatin to the cells cultivation, in three different concentrations, cells proliferation, cells cycle and the growth factors were evaluated in 24, 48 and 72 hours. The factors evaluated were interleukin 6, the growth factor of the vascular endothelium, the proliferation factor of fibroblasts and the transformation β factor. They influence in the angiogenesis, cell proliferation, metastasis formation and in the tumorous cells answers to the chemotherapy agents. There was a reduction of the cell availability and cells accumulation in the phase G0/G1 of the cell cycle, and that these effects are more accentuated in higher pravastatin concentration and contact period. A reduction of factors FGF and VEGF has occurred. The results show that the effects provided by pravastatin could be beneficial to patients with MM and there can be perspectives for additional studies to be performed, proving the benefits and safety. / O mieloma múltiplo (MM) atinge células plasmáticas completamente diferenciadas, que produzem elevadas quantidades de imunoglobulinas. As alterações genéticas são múltiplas e complexas, afetando a sensibilidade das células tumorais às drogas empregadas e o decurso da doença. A medula óssea constitui o microambiente ideal para progressão da doença, secretando fatores de crescimento imprescindíveis para a proliferação, sobrevivência e migração das células tumorais. Neste trabalho buscou-se avaliar os efeitos da pravastatina, droga inibidora da enzima HMG-CoA reductase, em células de MM, linhagem RPMI 8226. As células foram mantidas em cultura, em meio RPMI, acrescido de 10% de soro fetal bovino, à temperatura de 37°C com atmosfera de 5% de CO2. Após adição da pravastatina ao cultivo celular, em três diferentes concentrações, avaliou-se a proliferação celular, ciclo celular e níveis dos fatores de crescimento em 24, 48 e 72 horas. Os fatores avaliados foram interleucina, Fator de crescimento endotelial, fator de proliferação dos fibroblastos e Fator de transformação β, que influenciam na angiogênese, proliferação celular,metastização e na resposta das células tumorais aos agentes quimioterápicos. Houve uma redução da proliferação celular e acúmulo de células na fase G0/G1 do ciclo celular, sendo que estes efeitos são mais acentuados em concentrações mais elevadas de pravastatina e tempo de contato maior. Ocorreu uma redução dos fatores FGF e VEGF. Os resultados mostram que os efeitos por pravastatina poderia ser benéfico para os pacientes com MM e abrem perspectivas para realização de novos estudos, comprovando seus benefícios e segurança.

Oxisteróis são derivados oxigenados do colesterol que podem ser formados por autoxidação ou por atividade de enzimas específicas. Eles apresentam inúmeras atividades biológicas, incluindo inibição da proliferação celular e citotoxicidade. Dentre os oxisteróis, 7-cetocolesterol (7-KC), que difere do colesterol por apresentar um grupamento cetônico no carbono 7, é conhecido por induzir morte celular por diferentes vias. Neste projeto de pesquisa pretendemos avaliar o uso de uma emulsão lipídica contendo oxisterol (OXLE) como veículo para entrega de 7-KC a células tumorais. Os efeitos tóxicos de OXLE foram avaliados nas linhagem celulares RPMI 8226 (mieloma múltiplo) e B16F10 (melanoma). As células foram tratadas com: a) uma emulsão conhecida por atuar funcionalmente como a LDL (LDE); b) OXLE (75 uM até 225 uM de 7-KC); c) 7-KC (100 uM). As distribuições das células nas diferentes fases do ciclo de duplicação celulares foram analisadas por citometria de fluxo com iodeto de propídio. Todos os tratamentos, nestas concentrações, levaram a acúmulo das células de mieloma na fase proliferativa do ciclo celular. Para analisar a resposta apoptótica, nos utilizamos o fluorocromo JC-1, que mede o potencial transmembranar mitocondrial. Ambas a linhagens celulares, quando tratadas com OXLE, apresentaram hiperpolarização do potencial transmembranar associado com progressivo decrescimento do mesmo. Os resultados mostram que os grupos OXLE e 7-KC induziram a uma morte massiva das células de mieloma. OXLE induziu um efeito citostático nas células de melanoma, caracterizadas por alterações de morfologia, com um aumento na polimerização das fibras de actina, hiperpolarização seguida de perda do potencial transmitocondrial (apoptose associada à despolarização) , presença de vacúolos autofágicos como figuras de mielina (morte celular programada do tipo II - Autofagia); e impossibilitando as células de fazer a citocinese, determinado pelo aparecimento de uma população de células poliplóides. Nossos resultados indicam que esta nova emulsão contendo um oxisterol em sua formulação não é apenas um veículo para moléculas hidrofóbicas, mas pode atuar como agente cistotático/citotóxico. A morte mediada por OXLE possui características de apoptose e autofagia. Estes resultados são promissores e o possível use de OXLE in vivo necessita de futuras investigações. / Oxysterols are oxygenated derivatives of cholesterol that may be formed by autoxidation or by action of specific enzymes. They exhibit a number of biologic activities including inhibition of cellular proliferation and cytotoxicity. Among oxysterols, 7-ketocholesterol (7-KC), that differs form cholesterol by a functional ketone group at C7, is known to induce cell death by different ways. Herein we evaluated the use of an oxysterol-containing lipid emulsion(OXLE) as a vehicle to deliver 7-KC to tumor cells. OXLE was evaluated regarding its toxicity on RPMI 8226 (multiple myeloma) and B16F10 (melanoma) cell lines. Cells were treated with: a) an emulsion known to act functionally as LDL (LDE); b) OXLE (75 uM to 225uM of 7KC); c) 7KC (100uM). The distribution of cells in the different phases of cell cycle was analyzed by flow cytometry with propidium iodide. All treatments, according to the concentrations, led the myeloma cells to an arrest in the proliferative phases of the cell cycle. To analyze the apoptotic response, we then used a fluorochrome (JC-1), which measures the mitochondrial transmembrane potential. Both cell lines when treated with OXLE had a hyperpolarization of the transmembrane potential that decrease progressively. The results showed that exposure to either OXLE or 7-KC induced a massive death of myeloma cells. OXLE had an cytostatic effect on melanoma cells characterized by morphologic alterations as increased polymerization of actin fibers; hyperpolarization followed by a dissipation of the mitochondrial transmembrane potential (apoptosis-associated depolarization); presence of IX autophagic vacuoles as myelin figures (programmed cell death type II - Autophagy); and impaired cytokinesis, as determined by the emergence of a polyploid population. Our results indicated that this new oxysterol-containing emulsion is not only a carrier for hydrophobic molecules, but it can act itself as a cytostatic/cytotoxic agent. Cell death mediated by OXLE have both apoptosis and autophagy characteristics. These results are promising and the possible use of OxLE in vivo warrants further investigation

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130. / QC 20100920

Το πολλαπλούν μυέλωμα είναι μια νεοπλασία η οποία, έως και σήμερα, παραμένει ανίατη. Στο ΠΜ, το ανοσοποιητικό σύστημα δε κατορθώνει να καταστρέψει τα κακοήθη πλασμοκύτταρα, ενώ υπάρχουν ενδείξεις ότι τα κύτταρα του όγκου παίζουν ενεργό ρόλο σε αυτό. Στην παρούσα διατριβή, ελέγξαμε ποικίλλες μυελωματικές σειρές σχετικά με την έκφραση των προ- και αντι-αποπτωτικών γονιδίων. Βρήκαμε ότι τα μυελωματικά κύτταρα εκφράζουν έναν μη φυσιολογικό φαινότυπο (fas high/bcl low). Μελετώντας τα κύτταρα του μυελού των οστών ενός ασθενούς με ΠΜ τύπου IgG/k σταδίου ΙΙΙΑ, διαπιστώσαμε ότι αυτά τα κύτταρα εξέφραζαν τα γονίδια granzyme B και perforin, τα οποία υπό κανονικές συνθήκες εκφράζονται μόνο από τα κυτταροτοξικά Τ λεμφοκύτταρα (CTLs) και τα φυσικά φονικά κύτταρα (ΝΚ). Προτείνουμε ότι πιθανόν το παραπάνω γεγονός αποτελεί έναν επιπλέον αμυντικό μηχανισμό των κυττάρων του όγκου εναντίον των CTLs και ΝΚ κυττάρων. Συγκεκριμένα υποθέτουμε ότι τα κύτταρα του όγκου προσελκύουν τα CTLs και τα ΝΚ κύτταρα και τα καταστρέφουν με τη διαδικασία της κυτταρόλυσης. Τα διφωσφονικά οξέα χρησιμοποιούνται ευρέως για τη θεραπεία του ΠΜ, ενώ η θαλιδομίδη χρησιμοποιείται σήμερα για τη θεραπεία πολλών τύπων ΠΜ. Μελετήσαμε την επίδραση της θαλιδομίδης, του ζολεδρονικού οξέος και το συνδυασμό τους στην επιβίωση των μυελωματικών κυττάρων. Βρήκαμε ένα συνεργιστικό αποτέλεσμα των δύο αυτών ουσιών. Όταν η θαλιδομίδη συγχοηγηθεί με το ζολεδρονικό οξύ σε ελάχιστους μη δραστικούς μηχανισμούς, μειώνει την τοξική επίδραση 50% του τελευταίου 3-4 φορές. / Multiple Myeloma (MM) is a plasma cell neoplasia which, to this day, remains incurable. In MM, the immune system fails to destroy the malignant plasmocytes, and evidence exists that the tumor plays an active part in this. In the present study, we tested various MM cell lines for the expression of pro- and anti-apoptotic genes. We found that MM cells express an abnormal phenotype, namely fas high/bcl low. By studying bone marrow cells from a patient suffering of MM IgG/k type stage IIIA, we found that those cells expressed granzyme B nad perforin, normally expressed by cytotoxic T cells (CTLs) and natural killer (NK) cells. We propose that this may constitute an additional acquired mechanism by the tumor cells to protect themselves against the host.

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.