Orientador: Patricia Moriel / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:28:35Z (GMT). No. of bitstreams: 1 Tuan_BrunaTaliani_M.pdf: 5204136 bytes, checksum: aae37ded0ab8bc9e6059c1b65b9860dd (MD5) Previous issue date: 2014 / Resumo: A cisplatina (CDDP) é um agente antitumoral potente e citotóxico, indicado para o tratamento de carcinoma de células escamosas de cabeça e pescoço (CCECP), concomitante a radioterapia. Seu uso está limitado devido ao surgimento de intensos efeitos adversos, como exemplo a nefrotoxicidade. O objetivo deste trabalho foi avaliar o estresse oxidativo antes e após a quimioterapia com CDDP (fase 1), e correlacioná-lo com a excreção de CDDP na urina (fase 2), avaliando a nefrotoxicidade. Foi realizado um estudo clínico observacional, longitudinal prospectivo, quantitativo, com amostragem consecutiva, realizado de agosto de 2011 a dezembro de 2013 no Hospital de Clínicas/UNICAMP. Foram incluídos sujeitos de ambos os sexos, entre 18 e 80 anos, com diagnóstico de CCECP, em seu primeiro dia (caso novo) no Ambulatório de Oncologia Clínica, que receberam como conduta terapêutica 3 ciclos de quimioterapia com altas doses de CDDP e radioterapia concomitante. Os sujeitos foram acompanhados pela equipe de farmácia clínica no dia do caso novo, em todos os retornos com a equipe médica (pré quimioterapia, após os 3 ciclos de quimioterapia), como também nos dias das quimioterapias. Foram investigados biomarcadores de estresse oxidativo (através dos testes de TBARS, FOX-2 e Nitrito) e excreção de CDDP (0-12, 12-24 e 24-48 horas após a quimioterapia), ambos na urina, como também a nefrotoxicidade (variação de creatinina, Cr e clearance de creatinina, ClCr). Para todas as análises considerou-se significativo p < 0,05. Na fase 1 (n = 33) os sujeitos apresentaram idade média de 55 anos, sendo a maioria homens, brancos, tabagistas e etilistas acentuados, com tumores localizados na faringe, e com pico de estresse oxidativo no período 0-12h após a administração de CDDP. A excreção de CDDP também apresentou maiores valores no período 0-12h, evidenciando uma relação entre eles. Pode-se observar que houve relação entre o período basal e 0-12h dos testes de FOX-2 e nitrito, demonstrando que após a administração de CDDP existe aumento do estresse oxidativo, porém este não se relaciona com os parâmetros de nefrotoxicidade. Na fase 2 (n= 95) os sujeitos apresentaram características semelhantes à fase 1, e não houve relação significativa entre estresse oxidativo, excreção de CDDP e parâmetros de nefrotoxicidade. Portanto este estudo sugere que existe o aumento de estresse nitrosativo e oxidativo após a administração de CDDP em pacientes com câncer de cabeça e pescoço / Abstract: Cisplatin (CDDP) is a potent cytotoxic and anticancer agent, indicated for the treatment of head and neck squamous cell carcinoma (HNSCC), concomitant radiotherapy. Its use is limited owing of severe side effects, mainly nephrotoxicity. The aim of this study was to evaluate oxidative stress before and after chemotherapy with CDDP (phase 1), and correlate it with urinary excretion (phase 2), and nephrotoxicity of CDDP. An observational clinical study, prospective longitudinal, and quantitative, with consecutive sampling during August 2011 to December 2013 at the Hospital de Clínicas/UNICAMP was performed. We included patients of both sexes, between 18 and 80 years, diagnosed with HNSCC, on his first day (new case) at the Clinical Oncology, who received a therapeutic conduct of 3 cycles of chemotherapy with high-dose cisplatin concomitant radiotherapy. The patients were followed up by clinical pharmacy staff in the new case and all returns with the medical team (pre chemotherapy, after 3 cycles of chemotherapy), and in the days of chemotherapy. Biomarkers of oxidative stress (using the TBARS test, FOX-2 and nitrite in urine) and excretion of CDDP (0-12, 12-24 and 24-48 hours after chemotherapy), both in urine, were investigated, as well as nephrotoxicity (creatinine variation, Cr and creatinine clearance, CrCl). A p value less than 0.05 were considered significant. In phase 1 (n = 33) patients had a mean age of 55 years, mostly white men, smokers and alcoholics accented with tumors located in the pharynx, and with a increased in oxidative stress in 0-12h after administration of CDDP. The excretion of CDDP also showed higher values during 0-12h, showing a relationship between them. It can be seen that there was a relationship between the baseline and the 0-12h of FOX-2 and nitrite test, indicating that after administration of CDDP there is increase in oxidative stress, but this is not related to the parameters of nephrotoxicity. In phase 2 (n = 95) patients showed characteristics similar to phase 1, and there was no significant relationship between oxidative stress parameters, excretion of CDDP, and nephrotoxicity. Therefore, this study suggests that there is an increase of nitrosative and oxidative stress after administration of cisplatin in patients with head and neck cancer / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Prostate cancer is one of the most common cancer-related causes of death among men. Chemotherapy is mainly used for treatment of its later stages, accompanied by unpleasant side effects. So far, the treatment of advanced stages of prostate cancer has not been sufficient, and new more effective alternatives are needed. The application of the TRAIL cytokine, which induces apoptosis in tumor cell, but is not toxic to nonmalignant cells, seems to be a promissing approach. However, TRAIL-based therapy is often limited by the emerging cancer cell resistance. Overcoming the resistance can be achieved by combination therapy of TRAIL with effective sensitizers. Within this work, a combination of TRAIL with platinum-based chemotherapeutic drugs such as cisplatin or its novel derivative LA-12 was applied in order to facilitate the elimination of prostate cancer cells. In the experimental part of this work, using Western blot and flow cytometry analysis it was shown that TRAIL in combination with CDDP or LA-12 effectively enhanced apoptosis in three human prostate cancer cell lines. This effect was accompanied with increased activation/amount of several proapoptotic Bcl-2 family proteins, while no changes in the level of the receptors for TRAIL were observed. These results demonstrated that especially the combination...

Aim of this thesis is voltammetric study influence of liposomal platinum cytostatics on cancer cells. One of the goals is summarize available informations about influence of cisplatine on cancer cells, its encapsulation into liposome and affection of this cytostatic cisplatin encapsulated in liposome on cancer cell lines. In literary recherche is detail description of these issues. Than is there specification of voltammetric methods, which serve to electrochemical detection of cisplatin. Based on literary recherche was chosen the best method for detection and subsequently the method was optimalized and than was applied to measuring itself.

Cancer cells are able to adapt to different stress factors such as hypoxia, which is caused by insufficient tumor vascularization. An increased acetylation status of histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines was found to be a mechanism of adaptation of these cells to hypoxia. An increase in acetylation of histones H3 and H4 is suggested to cause changes in the structure of chromatin that lead to activation of gene transcription. In addition, cultivation of tested neuroblastoma cells under hypoxic conditions changes expression of proteins of a transcription factor N-myc, which is essential for development of neuroblastomas. This transcription factor is also responsible for a metabolic adaptation of neuroblastoma cells, increases their aggressiveness and its expression leads to a worse prognosis of the disease. Inhibitors of histone deacetylases (HDAC) are suggested to be the promising agents exhibiting various anticancer effects. They can induce cell cycle arrest, differentiation or programmed cell death in sensitive tumors. In this study, the effect of one of inhibitors of HDACs, valproate, on expression of proteins of transcription factors N-myc and hypoxia inducible factor 1α (HIF-1α) was investigated. Valproate decreases protein levels of both transcription factors in...

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The main reasons for the limited clinical efficacy of the platinum(II)-based agent cisplatin include drug resistance and significant side effects. Due to their better stability, as well as the possibility to introduce biologically active ligands in their axial positions constructing multifunctional prodrugs, creating platinum(IV) complexes is a tempting strategy for addressing these limitations. Another strategy for developing chemotherapeutics with lower toxicity relies on the ability of nanoparticles to accumulate in greater quantities in tumor tissues through passive targeting. To combine the two approaches, three platinum(IV) conjugates based on a cisplatin scaffold containing in the axial positions derivatives of caffeic and ferulic acid were prepared and loaded into SBA- 15 to produce the corresponding mesoporous silica nanoparticles (MSNs). The free platinum(IV) conjugates demonstrated higher or comparable activity with respect to cisplatin against different human breast cancer cell lines, while upon immobilization, superior antiproliferative activity with markedly increased cytotoxicity (more than 1000-fold lower IC50 values) compared to cisplatin was observed. Mechanistic investigations with the most potent conjugate, cisplatin-diacetyl caffeate (1), and the corresponding MSNs (SBA-15|1) in a 4T1 mouse breast cancer cell line showed that these compounds induce apoptotic cell death causing strong caspase activation. In vivo, in BALB/c mice, 1 and SBA-15|1 inhibited the tumor growth while decreasing the necrotic area and lowering the mitotic rate.

The application of ICP-MS to the fields of proteomics and genomics has arisen in part due to its ability to detect and quantify trace levels of S and P, which are major constituents in proteins and nucleic acids respectively. The development of collision/reaction cell technology and high resolution instruments has enabled these biologically important elements to be measured and quantified at the pg - ng ml-1 level. Despite these advances, the detection limits of P and S are still inferior compared to other elements. Oligonucleotides containing biotin functionality were labelled with Au nano-particles attached to a streptavidin protein to achieve site specific labelling, with 100% labelling efficiency. Each nano-particle contained ~86 Au atoms, resulting in an 882 fold signal enhancement for 24 base length oligonucleotides. However, this enhancement factor was only observed when one oligonucleotide bound to one nano-particle in a 1:1 ratio. Much lower Au labelling efficiencies and signal enhancements were observed when thiolated oligonucleotides were labelled with maleimide functionalised gold nano-particles. This was attributed to the extensive and difficult sample preparation steps that were required prior to labelling. The detection and quantification of adducts formed between DNA and the Pt anti-cancer drugs cisplatin and oxaliplatin were also investigated with ICP-MS. Acid digestion of the carbon based DNA matrix enabled Pt adducts to be quantified at low dose rates of 1 Pt atom per 1 500 000 nucleotides in ~12 μg DNA. Such sensitive mass spectrometric determinations could be employed in clinical tests to detect and quantify low level adducts formed in patients in-vivo. To complement ICP-MS analysis, electrospray ionisation linear ion trap mass spectrometry was employed to study the interaction of oxaliplatin with the four DNA nucleobases. Multiple stage mass spectrometry enabled detailed Pt-nucleobase adduct fragmentation pathways to be established. The method of DNA detection using P in conjunction with the collision cell, or cool plasma to form PO+ was also demonstrated and the limitations of the method, namely, polyatomic interferences and severe matrix effects were highlighted.

There is a significant current need to elucidate the molecular mechanisms of the side-effects caused by widely-used pharmaceuticals. Examples include the acute nephrotoxicity and irreversible ototoxicity promoted by the cationic drugs gentamicin and cisplatin. Gentamicin is an aminoglycoside antibiotic used for the prevention and treatment of life-threatening gram-negative bacterial infections, such as tuberculosis and meningitis. Cisplatin is used to treat a broad spectrum of cancers including head and neck, ovarian, cervical, stomach, bladder, sarcoma, lymphoma, testicular cancer and others. The objective of this study is to design and synthesize rhodamine derivatives that can be used for the construction of geometrically well-defined cationic drug conjugates. The long-term goal is to use the conjugates as tools to aid in elucidating the properties and identities of ion channels involved in the uptake of cationic pharmaceuticals into kidney and cochlear hair cells. This will shed light on the origin and potential prevention of unwanted side effects such as nephrotoxicity and ototoxicity associated with specific cationic drugs. A series of extended rhodamine analogs with reactive groups for biomolecule conjugation has been synthesized. These fluorophores show similar spectral properties to their prototype, Texas Red succinimidyl ester (TR-SE). However, they contain rigid linkers between the fluorophore and amine-reactive moiety. The resultant gentamicin conjugates of these materials are rigidified enabling one to assess channel pore dimensions without the confounding issue of conjugate folding. Preliminary cell studies are promising, as one observes reduced gentamicin uptake in both kidney and sensory hair cell upon systematically increasing the dimension of the fluorophore. This work has enabled us to tentatively assign the maximum dilated MET channel pore size as between 1.44 nm to 1.56 nm. However, this preliminary finding, though encouraging, needs further validation via ongoing studies with larger diameter fluorophore conjugates, A cisplatin-Texas Red conjugate has also been synthesized to enable studies of cellular uptake mechanisms. This conjugate preserves not only the spectral properties of Texas Red after conjugation, but also the cytotoxicity of cisplatin. This has been validated in zebrafish. The series of rhodamine probes that have been conjugated to gentamicin should be similarly useful for cisplatin studies. These studies are planned. Additional future work includes the synthesis of semi-flexible (glycol) and flexible (alkyl) linkers to evaluate structure-activity relationships.

Le cancer de l’ovaire est un cancer ayant un taux de décès particulièrement élevé. Les patientes répondent habituellement bien aux traitements chimiothérapeutiques mais la majorité connaîtront une rechute. Plusieurs mécanismes ont été identifiés comme partiellement responsables du développement de la résistance clinique à la chimiothérapie, dont la réparation plus efficace de l’ADN par la réparation par excision de nucléotides (NER). L'un des agents communément utilisés pour traiter ce cancer est le cisplatine, qui induit des dommages à l'ADN réparés par le NER. Une étude précédente de notre laboratoire a démontré qu'une déficience uniquement en phase S de la réparation par le NER peut se produire. Cette déficience est aussi dépendante de la kinase ATR. Nous avons choisi de déterminer si cette déficience est présente dans certains cas de cancer de l’ovaire et si cette déficience joue un rôle sur la résistance à la chimiothérapie. Nos objectifs sont donc : (i) vérifier la présence de cette déficience dans diverses lignées isolées du cancer de l’ovaire ; (ii) vérifier si le traitement chimiothérapeutique par des agents à base de platine peut favoriser la survie de cellules ayant une meilleure capacité de réparation par le NER; (iii) mesurer la sensibilité de ces lignées au cisplatine et vérifier si ceci corrèle avec leur capacité de réparation par le NER en phase S; (iv) déterminer si cette déficience est causée par la kinase ATR dans ces lignées. Nous avons déterminé qu’une déficience importante de la réparation par le GG-NER en phase S est présente dans de nombreuses lignées. De plus, des lignées isolées d’une même patiente pré-chimiothérapie et post-chimiothérapie montrent une augmentation significative de leur capacité de réparation par le GG-NER en phase S, suggérant un rôle de ce processus dans la résistance à la chimiothérapie. Nous avons aussi démontré qu’il y a une corrélation entre la capacité de réparation en phase S par le GG-NER et la sensibilité des lignées au cisplatine. Toutefois, nos résultats suggèrent que cette déficience n’est pas causée par ATR dans ces lignées puisque la phosphorylation de H2AX en réponse aux UV est similaire dans toutes les lignées. En plus d’un important apport fondamental, cette étude permettra d’étudier un potentiel mécanisme de résistance aux traitements chimiothérapeutiques dans le cancer de l’ovaire humain. / Ovarian cancer is one of the most lethal cancers. Patients usually respond very well to chemotherapy but most will eventually relapse. Many molecular processes have already been shown to influence chemotherapy resistance, including more efficient DNA repair by nucleotide excision repair. One commonly used drug for the treatment of ovarian cancer is cisplatin, a drug inducing lesions on DNA that can be repaired by NER. A previous study in our lab has shown that NER can be deficient specifically in S-phase. This effect is dependent on the kinase ATR. Thus, we chose to explore the possibility that this deficiency has an impact on resistance to cisplatin. Our objectives are: (i) to study the repair profile by NER in S-phase in ovarian cancer cell lines; (ii) to test if chemotherapeutic treatment can modulate nucleotide excision repair in cell lines by selecting cells that repair damage by NER more efficiently; (iii) to measure the sensitivity of these cell lines to cisplatin; (iv) to test if this deficiency can be attributed to ATR signalling. We show in this study that many ovarian cancer cell lines have an important defect in GG-NER specifically in S-phase. Pairs of cell lines that were isolated before and after chemotherapeutic treatment show an increase in their GG-NER efficiency in S-phase, suggesting a role for this enzymatic process in chemotherapy resistance. Also, we have found a correlation between the efficiency of GG-NER in S-phase and the sensitivity to cisplatin in the cell lines used in our study. However, the defect in GG-NER in S-phase in these ovarian cancer cell lines doesn’t seem to be due to ATR because the phosphorylation of H2AX in response to UV is equivalent between the different cell lines. This study will have an impact on our understanding of the fundamental aspects of DNA repair but could also provide insights on a potential novel mechanism of resistance to chemotherapy.