Thèses sur le sujet « Metronomica »

Il tumore alla mammella triplo-negativo (TNBC) rappresenta il sottotipo di tumore alla mammella più aggressivo ed è spesso associato ad una breve sopravvivenza libera da malattia. La dose massima tollerata, utilizzata come terapia standard (STD), rimane la prima opzione terapeutica per pazienti con TNBC, anche se non è sufficiente nel bloccare le recidive, le quali rimangono la prima causa di morte per questo tipo di tumore. Recentemente, è stato testato un diverso approccio terapeutico, chiamato chemioterapia metronomica (mCHT), che prevede la continua somministrazione della minima dose biologicamente attiva senza pause libere da farmaco. Ad oggi, i risultati mostrano una forte stabilizzazione della crescita del cancro ed un miglioramento della qualità della vita delle pazienti. La chemioterapia metronomica ha un effetto sia antitumorale che anti-angiogenico. In questa tesi abbiamo indagato, per la prima volta, l’effetto della somministrazione metronomica di 5-Fluorouracile (5-FU) e Vinorelbina (VNR) su linee cellulari di TNBC ed endoteliali, in confronto alla somministrazione STD. In particolare, abbiamo dimostrato che la somministrazione metronomica di 5-FU e VNR, sia in singolo che in combinazione, colpisce le cellule endoteliali e di TNBC a dosi significativamente inferiori della terapia STD. Nonostante le bassi dosi utilizzate, la somministrazione metronomica di 5-FU+VNR è più efficiente della STD nell’inibire la migrazione cellulare e la formazione di colonie sia delle cellule endoteliali che delle cellule TNBC. Per simulare l’interazione tra cellule endoteliali e TNBC, abbiamo utilizzato una co-coltura indiretta. I risultati della co-coltura indiretta mostrano che il terreno condizionato dalle cellule TNBC trattate con 5-FU+VNR secondo protocollo metronomico bloccano completamente la migrazione e la formazione di colonie delle cellule endoteliali. Inoltre, abbiamo evidenziato la dipendenza del meccanismo di morte cellulare delle cellule endoteliali e TNBC dalla modalità di somministrazione dei farmaci. In particolare, la combinazione metronomica promuove l’apoptosi nelle cellule endoteliali, mentre nelle cellule TNBC cambia la modalità di morte cellulare dall’apoptosi, indotta dalla terapia STD, all’autofagia, attivando anche la senescenza cellulare. In conclusione, la somministrazione metronomica di 5-FU+VNR è più efficiente della STD nel controllare la proliferazione, la migrazione e la capacità di ricrescita dopo trattamento delle cellule endoteliali e TNBC, suggerendo un miglior controllo sulla crescita tumorale e sulla formazione di recidive. Questi dati evidenziano inoltre i meccanismi molecolari alla base della migliore efficacia della terapia metronomica rispetto alla standard.<br>Triple-negative breast cancer (TNBC) represents the most malignant subtype of breast cancer, often associated with short disease-free survival. The maximum tolerated dose, used as standard-of-care chemotherapy (STD), remains the primary therapeutic option for TNBC patients, even though it fails to block distant recurrences, which is the first cause of death. Recently, it has been tested a different therapeutic approach, called metronomic chemotherapy (mCHT), where the minimum biological effective dose of the chemotherapeutic agent is given as a continuous administration without drug-free breaks. So far, the results show a strong stabilization of cancer growth and improved cancer patients’ quality of life. mCHT has direct cytotoxic effects on tumors and inhibits endothelial cell proliferation. In this thesis we investigated, for the first time, the effect of the mCHT administration of 5-Fluorouracil (5-FU) plus Vinorelbine (VNR) in endothelial and TNBC cell lines in comparison to the STD treatment. In particular, it has been shown that the mCHT administration of 5-FU and VNR both in single and in combination affects endothelial and TNBC cells at doses significantly lower than the STD one. Despite the low doses used, mCHT 5-FU+VNR is more effective than the STD in inhibiting cell migration and colony formation of both endothelial and TNBC cells. To simulate TNBC and endothelial cells' crosstalk, we utilized an indirect co-culture, which showed that medium conditioned by TNBC cells treated with mCHT 5-FU+VNR completely blocks endothelial cells migration and colony formation. We also pointed out that the modality of cell death of endothelial and TNBC cells depends on the schedule of treatment. In particular, the mCHT combination promotes endothelial’ apoptosis, whereas TNBC switches the modality of cell death from apoptosis, induced by STD, to autophagy, also activating senescence. In conclusion, mCHT 5-FU+VNR is more efficient than the STD in controlling cell proliferation, migration, and regrowth capacity after treatment of HUVECs and TNBC cells, suggesting better control of tumor growth and relapses. These data also highlight the molecular mechanisms that underlie the best efficacy of metronomic therapy towards the standard one.

Treatment strategies for advanced malignancy remain limited in their success, despite major advances in the understanding of cancer aetiology and molecular biology. The incidence of many cancers, including melanoma, continues to rise, with a huge demand for therapies even if treatment goals are purely cytostatic. One particular therapeutic strategy is the metronomic (continuous and low) dosing of conventional chemotherapy. There is evidence to suggest that tumour vasculature is the main target of this dosing schedule resulting in an overall 'non specific' anti-angiogenic effect. It is now being studied in clinical trials alone and in combination with specific anti- angiogenic agents. This thesis had two main aims: firstly to investigate the additive or synergistic anti- endothelial effects of a number of conventional cytotoxic agents (Temozolomide, Paclitaxel, Vinorelbine, Etoposide, Carboplatin) in vitro given in a metronomic schedule in combination with a specific anti-angiogenic compound (Sorafenib) and a non-specific sompound (Combretastatin). The anti-proliferative, cytotoxic activities of the metronomic combinatorial schedules were assessed on microvascular endothelial cells and cancer cells usmg an MTT proliferation assay. Results confirmed significant (p<O.OOl) endothelial-specific anti-proliferative effects induced by cytotoxics given at metronomic doses (e.g. Temozolomide at l Ou M, Paclitaxel at 1 25/-lM, Vinorelbine at InM). These anti-endothelial effects were significantly enhanced by the addition of sorafenib (p<O.OOl) in all except for with Vinorelbine, but were not enhanced by the addition of combretastatin. Secondly, the aim was to isolate circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) in patients with advanced (stage IV) melanoma. This was a scientific clinical sub-study of an on-going clinical trial to assess the efficacy of metronomically-dosed temozolomide and estramustine. After optimisation of the isolation techniques for CECs and CEPs from whole blood samples, the metronomically-dosed participants were compared to conventionally-dosed participants. This required an appropriate MREC amendment to the already existing clinical trail. Although numbers were very small and the study was aimed at hypothesis - generation, results showed a trend towards increased CECs mid- treatment in the metronomic group (mean: 6.6 CEC/IlL of whole blood pre-treatment versus 11.25 CEC/IlL of whole blood mid-treatment). There was otherwise no significant difference between the two groups. In summary, this thesis is a study of a combinatorial strategy to enhance the anti- angiogenic effects of the metronomic approach. This is a generic 'cancer treatment' approach, but is studied here more specifically in the setting of melanoma. It shows successful further development of an in vitro anti-endothelial assay which can potentially be used as a preclinical screening tool to determine efficacy of numerous agents and combinatorial regimens. It also demonstrates the isolation of potential biomarkers for anti-angiogenic therapy (i.e. CECs and CEPs) in a small but unique subset of patients with stage IV malignant melanoma.

In the present study the mechanisms leading to acquired chemoresistance, as well as new treatment strategies implying the prevention evading of tumor cells were addressed. Resistance formation is one of the major hurdles in cancer therapy. Metronomic antiangiogenic treatment of xenografted prostate cancer tumors in mice with cyclophosphamide (CPA) results in the appearance of resistant tumors. To investigate the complex molecular changes occurring during resistance formation, a comprehensive gene expression analysis of the resistant tumors in vivo was performed. A multitude of differentially expressed genes, e.g. PAS domain containing protein 1 (PASD1), annexin A3 (ANXA3), neurotensin (NTS) or plasminogen activator tissue (PLAT), were observed, when comparing resistant to in vivo passaged tumor samples. Moreover, tumor cells from in vivo and in vitro conditions showed a significant difference in target gene expression. For clarification of the mechanisms leading to the survival of tumor cells during maintained anti-angiogenic CPA therapy the differentially expressed genes were assigned to functional pathways like: axon guidance, steroid biosynthesis and complement and coagulation cascades. As blood flow might play a crucial role during maintained anti-angiogenic therapy, further analysis was focused on the genes grouped in complement and coagulation cascades. pregulation of anti-coagulatory ANXA3 and PLAT and downregulation of SERPIN A1 and other SERPIN-family members was shown by qPCR analysis. In contrast coagulation factor F3 was upregulated, accompanied by the expression of an altered gene product. Taken together, a potential role of anticoagulation as a resistance mechanism for anti-angiogenic CPA therapy could be described. Furthermore, the role of archazolid, a novel myxobacterial V-ATPase inhibitor in cancer treatment and in particular its action on the secreted cellular proteome was evaluated. As extracellular protein secretion may have an impact on invasive properties of tumor cells, the changes of the secretome profile of highly migratory urinary bladder carcinoma cells upon archazolid treatment were analyzed. An induced secretion of prometastatic lysosomal proteins such as the cathepsin family was observed. Interestingly, intracellular cathepsin B activity however strongly decreases and mature cathepsin B protein diminishes. It could be shown that archazolid inhibits the mannose-6-phosphate receptor mediated trafficking of procathepsin B from the trans-Golgi network to prelysosomal compartments, leading to an impaired cathepsin B maturation process. This results in an unnatural secretion of the inactive proenzyme and a dramatic decrease in intracellular cathepsin B activity. Importantly, also in vivo an archazolid induced reduction of cathepsin B activity was proven and archazolid treatment resulted in a reduced formation of distant metastases in the lungs. In summary these results indicate that archazolid in addition to its known anti-migratory properties might exert an anti-metastatic effect by reducing the activity of pro metastatic proteases like cathepsin B.

Made available in DSpace on 2015-10-06T13:03:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-15. Added 1 bitstream(s) on 2015-10-06T13:18:49Z : No. of bitstreams: 1 000848999.pdf: 1544826 bytes, checksum: 3fb73afedffc7650ac4620d6a79c29e9 (MD5)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>The canine mammary tumor (CMT) is the neoplasia that most commonly affects not spayed bitches. Therapy for CMT is a challenge since there are not described effective treatments for high-grade tumors. A therapeutic option that slows down tumor angiogenesis is metronomic chemotherapy (MC). Quantification of tumor angiogenesis has been proposed by measuring the vascular endothelial growth factor (VEGF) and microvessel density (MVD). Thus, in this study, the response of canine mammary carcinomas by the MC was evaluated by the measurement of MVD and tissue VEGF detection, apoptotic index (AI) and cell proliferation index (PI). Twenty-eight dogs with malignant CMT, equally distributed into a control group (CG) treated with mastectomy and a treated group (TG) treated with MC (cyclophosphamide 15mg/m2 and piroxicam 0.3 mg/kg) both orally and daily for 28 days followed by mastectomy. Mammary tumors were classified and graded histologically. MVD, tissue VEGF, AI and PI of all malignancies were obtained by immunostaining. The analysis showed statistical difference between groups in MVD and AI (p <0.05), showing quantitative reduction in tumor microvasculature and increase in tumor cells apoptosis. Based on this result, we can affirm that MC has antiangiogenic and proapoptotic effects in mammary carcinomas of bitches and can be used as a new therapeutic option

Resumo:A neoplasia mamária canina (NMC) é o tipo de neoplasia que mais comumente afeta cadelas não castradas. A terapia para a NMC é um desafio já que não são descritos tratamentos efetivos para neoplasias de alto grau histológico. Uma possibilidade terapêutica que age diminuindo a angiogênese tumoral é a quimioterapia metronômica (QM). Tem sido proposta a quantificação da angiogênese tumoral através da mensuração do fator de crescimento endotelial vascular (VEGF) e da densidade microvascular (DMV). Desta forma, neste estudo, foi avaliada a resposta de carcinomas mamários caninos à QM por mensuração da DMV e detecção do VEGF tecidual, índice apoptótico (IA) e índice de proliferação celular (IP). Foram utilizadas 28 cadelas com carcinomas mamários, distribuídas igualmente em um grupo controle (GC) tratadas com mastectomia e um grupo tratado (GT) com QM (ciclofosfamida 15mg/m2 e piroxicam 0,3mg/kg) ambos por via oral e diariamente durante 28 dias seguido de mastectomia. As neoplasias mamárias foram classificadas e graduadas histologicamente. DMV, grau do VEGF tecidual, IA e IP de todas as neoplasias foram obtidas por imunomarcação. A análise estatística mostrou diferença entre os grupos na DMV e IA (p<0.05), evidenciando redução quantitativa na microvasculatura tumoral e aumento na apoptose de células neoplásicas. Com base neste resultado, é possível afirmar que a QM possui efeitos antiangiogênicos e pró-apoptóticos em carcinomas mamários de cadelas e pode ser utilizada como uma nova opção terapêutica<br>Abstract:The canine mammary tumor (CMT) is the neoplasia that most commonly affects not spayed bitches. Therapy for CMT is a challenge since there are not described effective treatments for high-grade tumors. A therapeutic option that slows down tumor angiogenesis is metronomic chemotherapy (MC). Quantification of tumor angiogenesis has been proposed by measuring the vascular endothelial growth factor (VEGF) and microvessel density (MVD). Thus, in this study, the response of canine mammary carcinomas by the MC was evaluated by the measurement of MVD and tissue VEGF detection, apoptotic index (AI) and cell proliferation index (PI). Twenty-eight dogs with malignant CMT, equally distributed into a control group (CG) treated with mastectomy and a treated group (TG) treated with MC (cyclophosphamide 15mg/m2 and piroxicam 0.3 mg/kg) both orally and daily for 28 days followed by mastectomy. Mammary tumors were classified and graded histologically. MVD, tissue VEGF, AI and PI of all malignancies were obtained by immunostaining. The analysis showed statistical difference between groups in MVD and AI (p <0.05), showing quantitative reduction in tumor microvasculature and increase in tumor cells apoptosis. Based on this result, we can affirm that MC has antiangiogenic and proapoptotic effects in mammary carcinomas of bitches and can be used as a new therapeutic option<br>Orientador:Alexandre Lima de Andrade<br>Banca:Sabryna Gouveia Calazans<br>Banca:Maria Cecilia Rui Luvizotto<br>Mestre

Dissertação de Mestrado Integrado em Medicina Veterinária<br>A quimioterapia metronómica (QM) consiste na administração de baixas doses de compostos citotóxicos, de forma contínua, sem pausa, por um longo período de tempo. O fármaco citostático mais utilizado em QM é a ciclofosfamida (CIC). Este estudo teve como objetivo avaliar os efeitos adversos induzidos pela QM com CIC, administrada por via oral, em cães e gatos, diagnosticados com neoplasias malignas, através da caracterização dos efeitos secundários manifestados. Incluiram-se todos os cães e gatos, machos e fêmeas, submetidos a tratamento quimioterápico de neoplasias malignas espontâneas, com doses baixas de CIC, tanto em monoterapia como em terapia combinada (meloxicam, piroxicam e toceranib) no Hospital Veterinário Berna, desde de março de 2014 a março de 2016. Verificou-se que 26 em 36 animais ocorreu pelo menos um efeito adverso relacionado com a QM, correspondente a 72,2% da amostra. Foram observados 101 efeitos adversos, o que correspondeu a 40 episódios de toxicidade gastrointestinal, 30 de mielotoxicidade, 16 de toxicidade hepática, 13 de toxicidade renal e 2 de cistite hemorrágica estéril. Em 18 animais observou-se a ocorrência de mais do que um efeito secundário em simultâneo, ao longo do tratamento, correspondendo a 50% da amostra. No que diz respeito à gravidade dos episódios observados, a maioria destes foram ligeiros, de acordo com o Veterinary cooperative group – common terminology criteria for adverse events (VCOG-CTCAE, 2011), dado que 60,4% dos episódios classificaram-se em grau 1 e 27,72% em grau 2. Apenas a 8,91% e 2,97% dos episódios foi atribuída a classificação em graus 3 e 4, respetivamente.<br>ABSTRACT - Clinical profile evaluation of toxicity after oral administration of cyclophosphamide in metronomic chemotherapy regimen in dogs and cats - retrospective study of 36 cases - The metronomic chemotherapy (MC) consists in the administration of cytotoxic drugs, continuously, without interruption, using doses lower than those conventionally used. The most widely used cytostatic drug, in MC, is cyclophosphamide (CYC). This study aimed to evaluate the clinical presentation of toxicity induced by the MC with CYC, orally administered in dogs and cats diagnosed with malignant neoplasms, through the characterization of the manifested side effects. In this study were included all cats and dogs, males and females, from Hospital Veterinário Berna, since March 2014 until March 2016, undergoing chemotherapy treatment of spontaneous malignancies with low doses of CYC, either solely or in combination with other drugs (meloxicam, piroxicam and toceranib). It was found that 26 out of 36 animals experienced at least one episode of toxicity associated with MC, corresponding to 72.2% of the animals. 101 events of toxicity were observed, which corresponded to 40 episodes of gastrointestinal toxicity, 30 of myelotoxicity, 16 of hepatotoxicity, 13 of nephrotoxicity and 2 of sterile hemorrhagic cystitis. In 18 animals more than one event of toxicity occured simultaneously, throughout the treatment, corresponding to 50% of the animals. With regard to the severity of the adverse events observed, most of them were mild in accordance with Veterinary Cooperative Group - Common Terminology Criteria for Adverse Events (CTCAE VCOG - 2011), once 60.4% of the events were classsified in grade 1 and 27.72 % in grade 2. Only 8.91 % and 2.97 % of the episodes were classified as grades 3 and 4, respectively.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related death. Current available chemotherapeutic options are not curative due in part to their resistance to conventional therapies. We have generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of AFP- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. Low dose metronomic administration of cyclophosphamide (LDM-CTX) causes in this model complete regression of the tumor mass with a significant increase in survival (p<0.0001), and reduction in aberrant angiogenesis, IL-6 and TNFα (but not VEGF) levels, hyperproliferation, and alteration of normal liver parenchyma, compared to untreated animals. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, off-therapy mice developed rapidly tumor growth, which was still sensitive to LDM-CTX therapy. The capacity of developing hepatoshperes in vitro was drastically reduced upon LDM-CTX treatment, which resulted in selection of CD13+ cells, showing that these cells are particularly resistant to therapy. Co-treatment of the CD13-targeting drug bestatin with LDM-CTX resulted in a dramatic reduction in tumor burden. Therefore, our results demonstrate the therapeutic efficacy of administering LDM-CTX and targeting the residual CD13+ CSC-like population in these novel orthotopic HCC models, and strongly suggests that this therapy could be implemented in clinical trials.

Syfte: Syftet med denna kandidatuppsats är att förklara de positiva och negativa aspekter elit-friidrottare upplever av mobiltelefonens metronoma ljudstimulans påverkan under anaeroba intervaller, för en eventuell vidareutveckling.Metod: En applikation med grundläggande funktionalitet, som kunde användas vid testtillfället, utvecklades. 8 stycken elitfriidrottare som vid testtillfället hade topp 20 bästa resultat i sin gren, och ålder, i Sverige genomförde 6 anaeroba intervaller utifrån deras eget träningsprogram. Varannan intervall fick de hjälp av mobiltelefonapplikationen och varannan fick de springa som vanligt. Därefter genomfördes öppna intervjuer för att samla in deras upplevelser.Resultat: Applikationen påverkade testpersonernas upplevelser på fyra olika sätt. 1: Mindre ansträngande att springa med samma hastighet. 2: Motiverande vid utmattning. 3: Jämnare löprytm. 4: Försämring vid tappad synkronisering till den metronoma ljudstimulansen.Slutsatser: Applikationen gav mestadels positiva upplevelser hos testpersonerna och har därmed potential att utvecklas till en fullskalig applikation som riktar sig mot elitfriidrottare. För fortsatt-utveckling av applikationen verkar synkronisering till löpningen vara det viktigaste för att löparen ska få en positiv upplevelse.

This study examined the effects of music and metronomic beat on heart rate (HR). ratings of perceived exertion (RPE). and physical endurance in healthy females ages 18 to 30. Volunteers were screened via a health questionnaire and the Astrand Work Test on a Monark bicycle ergometer. yielding a predicted aerobic working capacity (V02) of 28 mililiters or more. The 30 subjects were then randomly assigned to one of two experimental conditions (music or metronome group) for a cycling test at 75% capacity. HR and Borg RPE were measured repeatedly, with a posttest measure of physical endurance (kilometers travelled). No significant differences were found between the groups in HR. RPE. or kilometers travelled. A music questionnaire administered to the music group following the posttest examined the subjects' preference for and familiarity with the music, perceived and preferred volume, and experience with music. Trends and suggestions for future research are discussed.

El treball descrit en aquesta tesi fa referència al tractaments de glioblastomes (GBM) GL261 preclínics que creixen en ratolins C57BL/6, també al seguiment no invasiu de la resposta a la teràpia, usant tècniques de ressonància magnètica (RM). El model immunocompetent GL261 GBM s’indueix per injecció estereotàctica de cèl·lules GL261 a l’estriat de ratolins WT C57BL/6. Tres agents terapèutics s’han provat en aquest model: el CX-4945®, inhibidor de la proteïna kinasa II (CK2), i dos agents alquilants d’administració oral usats habitualment en pràctica clínica pel tractament de GBM: la temozolamida (TMZ) i la ciclofosfamida (CPA). La CK2 ha estat descrita com a diana potencial pel tractament del càncer, ja que contribueix en el desenvolupament tumoral, proliferació i supressió de l’apoptosi. A més, nivells elevats d’expressió s’han demostrat en varis tipus de càncer. Malgrat això, el CX-4945, el qual està en fase clínica I/II, no va produir l’efecte beneficiós desitjat descrit per altres autors quan es va usar amb el nostre model de GBM GL261. A més, el tractament d’aquest model amb 3 cicles de TMZ, ja ha estat descrit en el nostre grup amb un augment significatiu de la supervivència. Per contra, la teràpia combinada de 3 cicles TMZ amb CX-4945, inesperadament, va revertir l’efecte beneficiós de la TMZ, el que va suggerir una interferència amb el sistema immunitari relacionat amb el desenvolupament i tractament del càncer. Això ens va portar a considerar l’ús d’un esquema de teràpia metronòmic (administració de dosis baixes separades per períodes iguals i sense llargs períodes de repòs entremig) descrit amb resultats prometedors a la literatura. La CPA, la TMZ i el CX-4945 van ser analitzats en un esquema de teràpia metronòmic a dosis diferents. Dins les diferents estratègies analitzades, els millors resultats es van obtenir amb el protocol metronòmic de teràpia combinada, cada 6 dies, de TMZ i CX-4945, mostrant un increment significatiu en la supervivència. Aquests resultats també van apuntar cap a la probable participació del sistema immunitari en resposta a la teràpia, tot i que es necessitaran futurs estudis d’histopatologia per confirmar aquesta hipòtesis. Altres resultats interessants addicionals van ser: en primer lloc, l’aparició d’un clar edema peritumoral durant moments concrets del tractament quimioterapèutic. En segon lloc, que la metodologia no-invasiva per determinar la resposta a la teràpia basat en l’anàlisi semi-supervisat de fonts d’imatge espectroscòpica de ressonància magnètica (MRSI), prèviament desenvolupat en el nostre grup amb ratolins tractats amb TMZ, també va demostrar ser viable per detectar la resposta induïda pel CPA en el nostre model preclínic. Això suggeriria que es pot observar un “patró metabolòmic de resposta” comú sota diferents estratègies terapèutiques. I en tercer lloc, l’aparició de limfomes trobats en les necròpsies de ratolins curats després de dosis cumulatives elevades de TMZ (480-1400 mg/Kg), suggerint que cal investigar estratègies per disminuir la dosi administrada d’agents alquilants per evitar efectes perjudicials en els ratolins tractats.<br>Work described in this thesis deals with the treatment of GL261 preclinical glioblastoma (GBM) growing in C57BL/6 mice, as well as with the non-invasive assessment of response to therapy using magnetic resonance (MR) techniques. The GL261 GBM is an immunocompetent model induced by stereotactic injection of GL261 cells into the striatum of C57BL/6 WT mice. Three different therapeutic agents have been tested in this model: CX-4945®, a protein kinase II (CK2) inhibitor, and two oral alkylating agents commonly used in the clinic for GBM treatment, temozolomide (TMZ) and cyclophosphamide (CPA). CK2 has been described as a potential suitable target for cancer treatment because it contributes to tumour development, proliferation, and apoptosis suppression in cancer. In addition, elevated CK2 expression levels have been demonstrated in several cancer types. Nevertheless, CX-4945, which already reached phase I/II clinical trials, did not produce the expected beneficial effect described by others when applied to our GL261 GBM model. Moreover, the GL261 GBM treatment with 3 TMZ cycles had been already described by our group with significant survival improvement. Nevertheless, the combined therapy 3 cycle-TMZ+CX-4945, unexpectedly, reverted the beneficial effect of TMZ, which suggested an interference with the immune cycle related with cancer development and treatment. This lead us to consider the use of a metronomic schedule (administration of low and equally spaced doses of drugs without long rest periods in between) described with promising results in the literature. CPA, TMZ and CX-4945 were assessed in a 6-day schedule metronomic schedule at different doses. Among the different strategies evaluated, best results were obtained with the combined metronomic administration, every 6 days, of TMZ and CX-4945 drugs, showing significant improved survival. This also pointed to the probable paticipation of the host mice immune system in therapy response, although further histopathological studies will be needed to fully confirm this hypothesis. Additional interesting findings were: firstly, a clear peritumoral brain edema appearance during certain stages of chemotherapeutic treatment. Secondly, that the non-invasive method for therapy response assessment based in semi-supervised source analysis of Magnetic Resonance Spectroscopy Imaging (MRSI) data, previously developed in our group with TMZ-treated mice, also proved useful for detecting CPA-induced response in our preclinical model. This would suggest that a common “metabolomics responding pattern” can be observed under different therapeutic strategies. And thirdly, the necropsy findings in mice cured from GL261 GBM after high TMZ cumulative dosage (480-1400 mg/Kg), which presented relevant lymphoma incidence, suggesting that strategies to decrease the administered dose should be investigated to avoid harmful effects in mice treated with alkylating agents.

碩士<br>國立臺灣大學<br>臨床動物醫學研究所<br>102<br>Metronomic chemotherapy, the low-dose, frequently administered chemotherapy has revealed an important impact on the stabilization of cancer disease. Metronomic chemotherapy has revealed recently as an alternative treatment protocol to slow down cancer progression by its known antiangiogenic effect and modulation of immune status without severe adverse effects. The aim of this study was to evaluate the treatment outcome, tolerability and safety of a combination low-dose chlorambucil at a dosage of 3-4 mg/m2 daily and standard-dose piroxicam at a dosage of 0.3mg/kg in spontaneous canine cancers. In addition, the effects of Treg percentages of peripheral blood had been test to determine the function of immune modulation. 21 dogs were enrolled in the study. The median progression-free interval and the median survival time were 140 days and 261 days. In macroscopic disease dogs, overall remission rate was 20% (two of 10). SD was noted in five of 10 dogs (50%). Microscopic disease, without metastasis at the presentation and low to intermediated clinical stage had a significant longer survival time. All the side effects mostly limited to the grade I-II gastrointestinal signs (anorexia, nausea/vomiting or diarrhea) without any hematological or urological toxicity. Only one dog (5%) had elevated creatinine level during treatment. The protocol was well tolerated without severe adverse effects. By series examination of Treg level, there were significant decreasing in mean percentage and absolute number of regulatory T cell of the 28 -day period for the response or stable dogs. In this study, metronomic chlorambucil with piroxicam revealed an acceptable antitumor effect in many kind of canine cancer without severe adverse effect.

碩士<br>國立中興大學<br>獸醫學系暨研究所<br>107<br>Metronomic chemotherapy (MC) is defined as the continuous oral administration of chemotherapeutic drugs at doses that are significantly lower than conventional maximally tolerated dose with no extended drug-free intervals. The mechanisms of MC included the effect on neovascularization, stimulating anticancer immune response and the induction of tumor dormancy. The aim of this study was to evaluate the clinical outcome of dogs with various malignant tumor treated with MC of cyclophosphamide (CTX) and/or piroxicam. This study was a retrospective study. One hundred and eighty-six dogs with malignant tumors and received CTX and/or piroxicam as MC were enrolled. The univariate analysis revealed that OST was significantly longer in the dogs treated with MC (CTX and/or piroxicam) greater than 28 days (P ＜ 0.001) and had undergone surgery (P ＜ 0.001) compared to dogs treated with MC (CTX and/or piroxicam) less than 28 days and without surgery. Furthermore, treated with MC (CTX and/or piroxicam) greater than 28 days and had undergone surgery were positively associated with significantly longer OST in dogs with distant metastasis. By multivariate analysis, treated with MC (CTX and/or piroxicam) greater than 28 days and had undergone surgery were the positive factors of longer OST. The overall response rate (including complete response or partial response) of MC (CTX and/or piroxicam) was 1.7%, while stable disease was noted in 58 dogs (33.3%). Two out of 186 dogs had grade 1 gastrointestinal toxicity. No grade 3 or 4 adverse effects were observed in dogs. Sterile hemorrhagic cystitis (SHC) occurred in 6 out of 186 dogs (3.2%). Median time to development SHC was 190 days. In conclusion, this study demonstrated that MC (CTX and/or piroxicam) treatment duration greater than 28 days and had undergone surgery had better outcomes in dogs with malignant tumors. Moreover, MC (CTX and/or piroxicam) was well-tolerated in dogs, but development of SHC should be concerned with dogs receiving MC (CTX and/or piroxicam).

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

Metronomic cyclophosphamide (CPA) treatment activates robust anti-tumor immunity and induces regression of implanted tumors in mouse models of brain cancer when administered on an intermittent, every 6-day schedule (CPA/6d), but not on a daily low-dose or a maximum-tolerated dose schedule. Five intermittent metronomic CPA schedules were investigated in GL261 gliomas implanted in scid mice. Metronomic CPA treatments spaced 9 or 12 days apart induced extensive tumor regression, however, tumor-infiltrating natural killer cell responses were not sustained, and tumor growth rapidly resumed after treatment day 24. Increasing the CPA dose prolonged the period of tumor regression on the every 9-day schedule, but natural killer cell activation was markedly decreased. Thus, sustained immune and anti-tumor responses were only achieved on the CPA/6d schedule. Furthermore, CPA/6d treatment eradicated GL261 tumors implanted in immunocompetent C57BL/6 mice by activating anti-tumor CD8-T cell responses and immune memory, which provides proof-of-concept that single agent chemotherapy delivered on an optimized metronomic schedule can cure large established cancers. Transcriptomic profiling, KEGG pathway, and upstream regulator analysis were employed to compare CPA/6d-induced gene expression changes between: immune-responsive GL261 tumors and immune-unresponsive Lewis lung carcinoma (LLC) and B16F10 melanoma tumors; between GL261 tumors implanted in immunocompetent mice versus in scid immunodeficient mice; and between GL261 tumors in scid mice treated with CPA every 6-days or every 9-days. CPA-treated LLC tumors were associated with inhibited VEGFA-targeted genes, down-regulated cell adhesion and transendothelial migration genes, and up-regulated drug metabolism pathways. In B16F10 tumors, CPA activated genes in chemokine signaling and antigen processing and presentation pathways, but no NK cell and T cell effector pathways were activated. GL261 tumors in scid mice were deficient in CPA activation of a subset of cytokine and cytokine receptor genes and T cell receptor signaling genes seen in immunocompetent mice. Cytokine gene expression was lower and drug metabolism gene expression was higher in every 9-day CPA-treated tumors versus CPA/6d-treated tumors. Together, these studies elucidate the dose, schedule, and adaptive immune-dependence of CPA-induced anti-tumor immune responses, giving new insight into the molecular signaling events underlying the deficiencies in immune responses seen in intermittent metronomic CPA-unresponsive tumor models.<br>2017-05-31T00:00:00Z

博士<br>國立陽明大學<br>傳統醫藥研究所<br>107<br>ABSTRACT Study background and aim Squamous cell carcinoma of the head and neck (SCCHN) is the 6th most common cancer and causes a major cancer-related mortality. In Taiwan, sixty-to-seventy percent of SCCHN arises from oral cavity, namely oral squamous cell carcinoma (OSCC). Despite combination treatments with surgery, radiotherapy (RT) and chemotherapy, the long term survival remains approximately 50%. Patients with recurrent or refractory diseases have even dismal prognosis with a median overall survival 7 to 10 months. There are great unmet medical needs in the OSCC treatment. Cordycepin, one of the important pure compounds purified from the fungal medicinal herb Cordyceps sinensis, possesses anticancer activity through a variety of mechanisms. The aim of our study was to investigate if cordycepin exerts anti-cancer effect against oral cancer through inhibiting the epithelial mesenchymal transition (EMT) and enhancing the radiation response of OSCC cells. Materials and Methods: OSCC cell lines, include SAS, OEC-M1 and OC-3 were used in the study. We performed MTT and colony formation assay to test the cytotoxicity and radiosensitizing effects of cordycepin. The cell cycle analysis was performed by flow cytometry. The apoptotic effect was measured by caspase 3 activity assay. The migration inhibition was demonstrated by wound healing assay. Hallmark of RT induced DNA double strand break (DSB), γ-H2AX, was testified by immunofluorescence and flow cytometry. The molecular modulation of EMT process and DNA damage response were evaluated by Western blotting. Lastly, the anticancer effect was examined in the OSCC-bearing mice model. Results: Cordycepin inhibited the OSCC cell viability in a dose and time dependent manner. Morphological characteristics of apoptosis and increased caspase 3 activity were observed. Cell cycle arrested on G2/M phase by treating with cordycepin alone. Combination of cordycepin and RT further prolonged G2/M arrest. Delayed tumor cell migration and inhibited EMT process were demonstrated by decreased E-cadherin and increased N-cadherin expression. This EMT inhibition was further examined by the immunohistochemical stain of the xenograft tumors. Colony formation assay revealed that cordycepin potentiated the RT cytotoxic effects. The combination treatment also prolonged the DNA DSB repair. We found that cordycepin decreased expression of ataxia telangiectasia-mutated kinase and checkpoint kinase 2 protein to prevent appropriate DNA repair and cell cycle arrest. Reciprocal upregulation in the expression of checkpoint kinase 1(Chk1) might suggest a compensatory cell protection mechanism. Furthermore, we demonstrated the enhanced radiosensitizing effect through siRNA knockdown of the Chk1 protein. Metronomic administration of cordycepin inhibited the OSCC-bearing mice tumor growth. Cordycepin combination treatment further augmented the growth inhibitory activity of RT. No major toxicities of the metronomic dosing were observed. Conclusion: In our study, we concluded that cordycepin possesses anticancer effects against OSCC cells through EMT inhibition and DNA damage response modulation. Development of cordycepin into a novel metronomic therapeutic agent as well as a radiosensitizer against OSCC needs further investigation.

Dissertação de Mestrado Integrado em Medicina Veterinária<br>A era da quimioterapia, iniciada a meio do século passado, tem sido dominada pelo uso habitual de protocolos de quimioterapia de dose intensa, os quais se baseiam no conceito de dose máxima tolerada. Ao promoverem um equilíbrio entre a qualidade de vida do animal e o objetivo de destruir prontamente o maior número possível de células tumorais, estes protocolos ocupam ainda hoje um lugar de destaque na prática clínica diária. Contudo, aquando da entrada do novo milénio, a quimioterapia metronómica foi introduzida como uma possível alternativa à utilização de quimioterapia convencional, trazendo consigo uma considerável mudança no panorama dos tratamentos quimioterápicos. Caracterizada por uma administração diária a longo prazo de doses mais baixas de fármacos citotóxicos, esta nova modalidade tem características completamente diferentes, além de benefícios animadores. Mas qual será o seu verdadeiro lugar no arsenal da terapia quimioterápica? Terá a quimioterapia metronómica eficácia suficiente para tratar animais com cancro? Ou será apenas uma opção paliativa e mais económica? Dentro da quimioterapia metronómica, uma nova terapia antitumoral tem emergido como a forma mais recente de administração de fármacos citotóxicos. O regime chemo-switch consiste na utilização de quimioterapia metronómica como uma terapia de manutenção, no seguimento da aplicação de quimioterapia convencional. No entanto, apesar de se apresentar como uma modalidade promissora, carece ainda, à semelhança da própria quimioterapia metronómica, de validação na prática clínica. O hemangiossarcoma canino é um tumor maligno com origem nas células endoteliais vasculares, cujo prognóstico é geralmente pobre, e o tratamento permanece particularmente desafiante em oncologia veterinária, devido ao seu altíssimo índice de metastização. Foram comparadas retrospetivamente as modalidades quimioterápicas existentes até à data (metronómica, convencional e o regime de chemo-switch) para o tratamento de 29 cães com hemangiossarcoma de estadio II-III. Os cães inseridos no grupo da quimioterapia metronómica receberam o protocolo CTP, que consistiu na combinação de três fármacos (ciclofosfamida, talidomida e piroxicam); dentro do grupo da quimioterapia convencional, os animais receberam o protocolo DOX (um protocolo de quimioterapia dose-intensa composto somente por doxorrubicina), ou o protocolo ADTIC (um protocolo de quimioterapia dose-intensa que combinava doxorrubicina com dacarbazina): o grupo do chemo-switch incluiu cães tratados com um dos dois protocolos de quimioterapia dose-intensa seguido do protocolo de quimioterapia metronómica. O estadio clínico destacou-se como sendo o único factor determinante para a análise de sobrevida dos animais, mais do que a própria abordagem terapêutica. Os cães diagnosticados com hemangiossarcoma de estadio II tiveram um tempo para a progressão mediano e uma sobrevida mediana superiores àqueles com estadio III (140 dias vs. 66 dias, P<0.001, e 198 dias vs. 87 dias, P<0.001, respetivamente). Por outro lado, não foi encontrada qualquer diferença estatisticamente significativa entre os três braços terapêuticos, nem quando o tempo para a progressão foi avaliado (P=0.985), nem para o tempo de sobrevida (P=0.906). É importante referir que todos os protocolos foram bem tolerados em termos de toxicidade e que, apesar de os resultados serem inferiores àqueles publicados na comunidade científica, o presente estudo retrospetivo preliminar pretende promover a elaboração de trabalhos prospetivos na área.<br>The era of chemotherapy, which started in the middle of the last century, has been ruled by the routine use of dose-intense protocols, based on the maximum-tolerated dose concept. By promoting a balance between patient’s quality of life, with the goal of rapidly killing as many tumor cells as possible, these protocols still occupy a prominent place in daily clinical practice. However, the opening of a new millennium has brought a considerable change in oncologic treatments, when metronomic chemotherapy was proposed as a possible alternative to the conventional drug delivery. Characterized by a long-term daily basis drug administration of shorter doses of cytotoxic drugs, this new modality has completely different features besides offering cheering benefits. But what is the real place of this strategy in chemotherapeutic arsenal? Will indeed metronomic chemotherapy have enough efficacy to treat animals with cancer? Or will it be just a palliative and cheaper choice? Inside metronomic chemotherapy, a new anticancer therapy has emerged as the most recent form of scheduling the administration of cytotoxic drugs. The so-called chemo-switch regimen consists in the use of metronomic chemotherapy as a maintenance approach, after the application of conventional chemotherapy. Despite being a promising modality, much like metronomic chemotherapy itself, the validation of chemo-switch remains to be verified in clinical practice. Canine hemangiosarcoma is a malignant tumor that arises from the vascular endothelial cells, which prognosis is generally poor, and treatment still particularly challenging in veterinary oncology, due to its very high metastatic rate. It was retrospectively compared the so far existent chemotherapeutic modalities (metronomic, conventional and the chemo-switch regimen) for the treatment of 29 dogs with stage II-III hemangiosarcoma. Dogs allocated into the metronomic group received the protocol CTP, which consisted in a combination of three drugs (cyclophosphamide, thalidomide and piroxicam); inside the dose-intense group, patients received the protocol DOX (a single-drug dose-intense protocol of doxorubicin), or the protocol ADTIC (a dose-intense doxorubicin-based protocol that combined doxorubicin with dacarbazine); the chemo-switch group included dogs that were treated with one of the two dose-intense protocols followed by the metronomic protocol. The clinical stage stood out as the only determining factor for the patients’ survival analysis, rather than the therapeutic approach itself. Dogs diagnosed with stage II hemangiosarcoma had a longer median time to progression and survival time than those with stage III (140 days vs. 66 days, P<0.001, and 198 days vs. 87 days, P<0.001, respectively). On the other hand, it was not found any statistically significant difference among the three treatment arms, neither when evaluating the time to progression (P=0.985), nor with the survival time (P=0.906). Importantly, all protocols were well tolerated in terms of toxicity, and even though the results were below the ones published in scientific community, this retrospective preliminary study intends to promote further prospective investigations in the field.