Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-19Bitstream added on 2014-06-13T18:51:13Z : No. of bitstreams: 1 oliva_cac_me_jabo.pdf: 178983 bytes, checksum: f310a85e1eb63818fa51dba6cafeadd8 (MD5) / A cisplatina é um fármaco antineoplásico utilizado como adjuvante no tratamento de diversas neoplasias. Neste estudo foram avaliados o hemograma e os teores de sódio, potássio, magnésio, cálcio e fósforo do soro sanguíneo de cães submetidos à terapia com cisplatina. Foram utilizados oito cães, machos, sem raça definida, com 10 a 15 kg de peso, clinicamente sadios. Os cães foram distribuídos em dois grupos, contendo 4 animais cada, sendo que os animais do grupo 1 receberam cisplatina e aqueles do grupo 2 não receberam cisplatina. Os cães do grupo 1 receberam quimioterapia e protocolo de diurese para proteção renal, já o grupo controle 2 não recebeu a cisplatina, estando sujeito apenas aos fatores ambientais. Os animais do grupo 1 foram submetidos a quatro sessões de quimioterapia com cisplatina na dose de 70mg/mø, administrada por via intravenosa, durante 20 minutos, no intervalo de 21 dias. Antes da administração da cisplatina, realizou-se fluidoterapia com solução fisiológica a 0,9% na dose de 25mL/kg/hora, por via intravenosa, durante duas horas, e depois por mais uma hora. Todos os animais receberam metoclopramida na dose de 2mg/kg, por via intravenosa, 15 minutos antes da administração da cisplatina e furosemida na dose de 2 mg/kg, por via intravenosa, 5 minutos após administração de metoclopramida. As amostras foram processadas e analisadas antes de cada sessão de quimioterapia. Os resultados mostraram que não houve diferença significativa entre os grupos para as contagens de hemácias, concentração de hemoglobina, hematócrito e contagem de leucócitos, mesmo assim as concentrações séricas de eletrólitos mantiveram-se dentro dos padrões da normalidade. Os resultados obtidos podem ser indicativos de que o protocolo empregado para o grupo 1 se mostrou efetivo para manter as características do hemograma e a concentração sérica dos eletrólitos. / The cisplatin is an antineoplasic drug used like adjunct treatment of various neoplasms. In this study, one evaluated the hemogram and sodium, potassium, magnesium, calcium and phosphorus levels in the dogs` blood under administration of cisplatin. One used 8 male dogs, with no definite race, weighing from 10 to 15 kilograms, and clinically healthy. The dogs were divided into two groups of 4 animals each, being group 1 treated with cisplatin and group 2 with no cisplatin. Group 1 received chemotherapy and the diurese protocol for kidney protection, group 2 did not receive cisplatin, being exposed only to the environmental factors. The animals from group 1 were submitted to four chemotherapy sessions with cisplatin 70mg/m2 administered intravenously for 20 minutes, in a 21 days interval before the cisplatin administration, one carried out a fluidotherapy with physiologic solution 0,9% on a dosage of 25mg/kg/hour intravenously during 2 hours, and posteriorly for one more hour. All the animals received methoclopramid intravenously on a dosage of 2mg/kg, 15 minutes before the cisplatin and furosemide administration on a 2mg/kg dosage, 5 minutes before the cisplatin infusion. The evaluation of the hemogram and the electrolytes levels above mentioned were done before each chemotherapy session. The results demonstrate that there were no significant differences among the groups for red blood cells counting, hemoglobin concentration, hematocrit and leucocytes counting, but still, the electrolytes seric concentration maintained itself in a normal standard. The results obtained may indicate that the protocol employed for group 1 showed efficiency to maintain the characteristics of the hemogram and the electrolytes seric concentration.

Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...

A cisplatina ainda é um dos agentes quimioterápicos mais efetivos. No entanto, em elevadas doses pode ocorrer hepatotoxicidade. Alguns antioxidantes têm sido mostrado amenizar a hepatotoxicidade induzida pela cisplatina mas o mecanismo molecular envolvido não está bem esclarecido.No presente estudo nós investigamos moleculares subjacente ao efeito protetor da dimetiltiuouréia (DMTU), um conhecido eqüestrador de radical hidroxil, contra a lesão oxidativamitocondrial hepática induzida pela cisplatina em ratos. Ratos Wistar machos adultos ( 200 a 220g) foram divididos entre 4 grupos de 8 animais cada. O grupo controle foi tratado apenas com uma injeção intraperitoneal (i.p.) de solução salina (1 ml/ 100g de peso). Ao grupo DMTU foi administrado apenas DMTU (500 mg/kg de peso, i.p., seguido de 125 mg/kg, i.p., duas vezes ao dia até o sacrifício). Ao grupo cisplatina foi administrado uma injeção única de cisplatina (10 mg/kg de peso, i.p.). Ao grupo DMTU + cisplatina foi administrado DMTU (500mg/kg de peso, i.p.), pouco antes da injeção da cisplatina (10 mg/kg de peso, i.p.), seguido por injeções de DMTU (125 mg/kg de peso, i.p.) duas vezes ao dia até o sacrifício ( 72 horas após o tratamento). A hepatotoxicidade foi evidenciada no grupo cisplatina pelo aumento dos níveis séricos de alanina (ALT) e aspartato (AST)aminotransferases. O mecanismo de hepatotoxicidade induzido pela cisplatina mostrou-se envolvido na rigidez de membrana; na redução da razão glutationa reduzida em relação a glutationa oxidada (GSH/GSSG); na redução dos níveis de ATP, GSH e NADPH; na lipoperoxidação; na lesão oxidativa da cardiolipina e de proteínas com grupos fidrílicos. Mais ainda, a morte celular por apoptose foi também demonstrada e os achados fortemente sugerem a participação do xi mecanismo sinalizador mitocondrial neste processo; o DMTU não apresentou nenhum efeito direto sobre a mitocôndria e inibiu substancialmente a lesão mitocondrial induzida pela cisplatina, prevenindo a hepatotoxicidade. Todos os seguintes efeitos induzidos pela cisplatina foram previnidos pelo DMTU: (a) elevação dos níveis séricos de AST e ALT; (b) redução dos níveis de ATP hepático;(c)peroxidação lipídica;(d)oxidação da cardiolipina; (e)oxidação de proteínas sulfidrílicas; (f) rigidez da membrana mitocondrial; (g) oxidação de GSH; (h)oxidação de NADPH e (i) morte celular por apoptose. Os resultados mostraram o papel principal da mitocôndria e dos radicais hidroxilas na proteção do fígado saudável contra a lesão hepática induzida pela cisplatina, delineando um número de etapas que podem ser consideradas no desenvolvimento de futuros agentes citoprotetores / Cisplatin is still one of the most effective chemotherapeutic agents. However, at higher doses hepatotoxicity may occur. Some antioxidants have been shown to ameliorate cisplatin-induced hepatotoxicity but the involved molecular mechanism has not been clarified. In the present study we investigated the molecular mechanism underlying the protective effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, against liver mitochondrial oxidative damage induced by cisplatin in rats.Adult male Wistar rats (200 to 220g) were divided into 4 groups of 8 animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1ml/100g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until sacrifice). The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 hours after the treatment). epatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatininduced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process; DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial injury, therefore preventing the hepatotoxicity. All the following cisplatin-induced xiv effects were prevented by DMTU: (a) elevation of AST and ALT serum levels; (b) decreased hepatic ATP levels; (c) lipid peroxidation; (d)cardiolipin oxidation; (e) sulfhydryl protein oxidation; (f) mitochondrial membrane rigidification; (g) GSH oxidation; (h) NADPH oxidation and (h) apoptotic cell death. Results show the central role of mitochondria and hydroxyl radicals in the protection of healthy liver against cisplatin-induced injury, highlighting a number of steps that might be considered in the development of novel cytoprotective agents.

Class of 2015 Abstract / Objectives: To compare and evaluate effects on kidney function of mannitol dieresis versus saline diuresis on kidney function with cisplatin therapy. Methods: Patient charts documented between January 2010 and July 2013 were obtained and reviewed from a database of a university associated medical center. The patient’s lowest creatinine clearance (CrCl) and potassium levels during any time in therapy were compared against the baseline. Statistical testing for primary and secondary outcomes was calculated using the Independent-Samples T-Test. Results: A total of 140 patients were reviewed – 68 patients were included in the mannitol arm, 72 in the saline arm. All baseline characteristics reviewed were not statistically different between groups except for sex, which was skewed towards males in the saline arm of the study. Baseline CrCl was 97.14 ml/min in the mannitol arm, and 93.69 ml/min in the saline arm (p=0.91). The average change in CrCl was found to be -16.72 ml/min (95% CI, -21.85 to -11.59) in the mannitol arm, -14.00 ml/min (95% CI, -18.82 to -9.20) in the saline arm; this was not statistically different (p=0.41). There was an average change of -0.31 mmol/L in blood potassium levels in mannitol patients, and a change of 0.014 mmol/L in saline patients; this was found to be significantly different (p<0.01). Conclusions: In this single-center retrospective study, there appeared to be no benefit in using mannitol diuresis over saline diuresis. The use of mannitol incurs additional cost and place additional restrictions on administration.

It is well established that many solid tumours are heterogeneous with respect to oxygenation, and contain regions of hypoxic cells, which due to their inherent resistance to ionizing radiation, limit the success of radiotherapy. Numerous chemicals and drugs have been investigated over the past few decades as potential radiosensitizers. The most notable of these being the organometallic compound, cis-diammine dichloroplatinum(II). The clinical success of this drug led to the synthesis of other types of organic cytotoxic metal-containing drugs. Prof. J.C. Swart from the University of the Orange Free State supplied seventeen novel iridium, ferrecenium and rhodium complexes, which I screened for cytotoxic activity against the CHO cell line. The two most cytotoxic complexes namely, [Rh(fctca)(cod)] and [Rh(fctfa)(cod)], were tested for radiosensitizing potential against aerobic and hypoxic CHO cells in the presence of an 8 MV photon beam by the MTT assay adapted to our laboratory conditions. The ferrocene betadiketones co-ordinated to them, Hfctca and Hfctfa and the Ir compliment of [Rh(fctfa)(cod)] namely, [Ir(fctfa)(cod)] were also assessed by the MTT assay. Interestingly, neither the ferrocene nor the iridium complexes showed noteworthy sensitization, which suggests that the rhodium is responsible for the efficacy observed. The radiosensitizing potential of the most active complex, [Rh(fctfa)(cod)] and cisplatin were also confirmed by the use of the more traditional clonogenic assay. Not only did the MTT assay deliver results comparable to the clonogenic technique, but one of the complexes [Rh(fctfa)(cod)] showed radiosensitizing potential against hypoxic CHO cells, equal to that of cisplatin. The rhodium complex, [Rh(fctfa)(cod)] was also tested for radiosensitization properties against the CHO cell line in the presence of a p(66)/Be neutron beam. Results indicated that [Rh(fctfa)(cod)] sensitizes cells to radiation possibly by inhibition of cell inactivation mechanisms that are normally associated with repairable damage. Consequent work done on the flow cytometer where direct DNA damage after irradiation (8 MV photon beam) and drug treatment, was assessed on aerobic CHO cells by a technique adapted to our laboratory showed no significant increase in the forward angle scattered light (FSC) parameter which is an indication of radiation induced strand breaks. Furthermore, [Rh(fctfa)(cod)] showed a significantly greater increase in the side angle scattered light (SSC) parameter, which is an indication of the binding ability of the complex, compared to cisplatin, after treatment with different concentrations of the drugs. Results obtained from enumerating micronuclei frequencies after drug treatment and radiation confirmed that both cisplatin and [Rh(fctfa)(cod)] are more active under hypoxic conditions, with [Rh(fctfa)(cod)] responsible for more micronuclei per binucleated cell. In conclusion, I have established that [Rh(fctfa)(cod)] has a cytotoxic activity comparable to that of cisplatin and that it sensitizes preferentially hypoxic CHO cells to radiation in the clinically relevant dose range. I have also identified the probable action by which [Rh(fctfa)(cod)] sensitizes CHO cells to radiation as being inhibition of repair capacity. Furthermore, results suggest that this complex binds covalently to DNA base pairs. The complex [Rh (fctfa) (cod)] , has so far proven to possess interesting radiosensitizing potential which must be exploited for eventual therapeutic benefit. / Dissertation (MSc (Medical Physics))--University of Pretoria, 2007. / Medical Oncology / unrestricted

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21997号 / 医博第4511号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 妹尾 浩, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Individual types of viability tests based on colorimetric changes of the solution are desribed in the theoretical part. Furthermore, HSP proteins are characterized, which are not connected only by heat shock, but also during other cell stresses such as exposure to UV, cold, extreme pH or heavy metals. They are important for the cell, because they help to reformulate proteins that have been damaged by cellular stress and also bind to new unpacked proteins and ensure their correct folding. Proteins that are affected by molecular chaperones are collectively called client proteins. Some HSPs also contribute to membrane transport or degradation. These proteins are co-operative with the cochaperones, which are important for heat shock proteins because they help them to pack protein, in particular by catalyzing the hydrolysis of ATP to ADP. Herein is also described cisplatin and its derivatives, including mechanism of action and adverse effects. This work was focused on detection cytotoxicity of cisplatin and its derivatives. Cells were exposed to stress condition induced by cytostatics and huge changes in heat shock proteins and cochaperon levels were observed. There was also observed colocalization of heat shock proteins and their client protein p53 by confocal microscopy in these stressing conditions.

Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...