The tumor suppressor protein p53 is one of the major regulators of the cell cycle after DNA damage. In addition to stopping the cycle and repairing DNA, it can, in extreme cases, induce programmed cell death - apoptosis. Mutations in the gene encoding p53 are present in more than 50% of cancer cases. This thesis examines alternative natural polyphenolic substances that could increase the level and expression of p53 protein in tumor cells. These substances could be an alternative to non-specific cytostatics, which bring many undesirable additional effects during treatment. In the theoretical part of the thesis the structure and properties of the p53 protein and describes alternative therapeutic approaches with a focus on polyphenolic substances is explained. The aim of the experimental part was to determine the effect of curcumin and resveratrol in comparison with often used cytostatic drug, doxorubicin, on cell viability of tumor cells and on p53 protein levels. The effect of these substances on the binding of p53 to DNA in yeast systems was also examined. It was found that doxorubin efficiency is many times higher than the examined polyphenolic agents, but resveratrol was showing some potential as a suitable alternative in the treatment of tumors, thanks to the ability to activate apotosis. It was clearly demonstrated that there is an association between induced programmed death and increased p53 protein expression after resveratrol treatment.

The theoretical part of the diploma thesis deals with tumour diseases and a general description of tumour suppressor genes. Special attention is paid to the TP53 gene and its influence on the functioning of the organism, including its production of the p53 protein. Based on an analysis and synthesis of findings from specialised sources, the current methods of detecting the aforesaid gene, in particular immunological and elec-trochemical, were described. The last chapter of the theoretical part is devoted to bio-sensors and their classification, with a special focus on electrochemical biosensors. The practical part involves the construction and optimisation of the biosensor, from the cleaning of electrodes, the deposition of gold nanoparticles, the ssDNA bond, the blocking of free binding points and a synthesis of the complementary chain to the resulting measurement and quality testing. This part also presents the concept of an innovative biosensor that offers an easier and more reliable alternative for the detec-tion of the TP53 gene. The last chapter of this part analyses and comments on the aforesaid results.

The present experiments were designed to evaluate the effects of pifithrin-α (PFT-α), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-α attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-α had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-α. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-α offered protection against all of the aforementioned changes. Finally, PFT-α did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-α effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-α has the potential to protect cancer patients against DOX-induced cardiac injury.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcription factor and tumor suppressor protein p53 critically regulates cell survival or death in response to cellular stress. p53 can activate genes involved in a wide variety of processes, including apoptosis, cell cycle arrest, angiogenesis, metabolism, and senescence. Mutations in p53 are common in cancer and alter its interactions with other proteins, but there are other mechanisms and posttranslational modifications that can alter these interactions as well. In some tumors, such as renal cell carcinoma, p53 is commonly inactive even though mutations to TP53 are rare. This suggests that there are other biochemical mechanisms of inhibition, which we explore in this study. Mutations in the DNA-binding domain of p53 result in conformational changes that enable p53 to interact with and inhibit its family member p73, thereby promoting cell survival instead of apoptosis. In contrast, it has been reported that wild-type p53 does not bind to p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53 to form a complex with p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes such as PUMA and BAX, as well as the induction of apoptosis. In addition to the apoptotic function of p53, the tumor suppressor also plays a major role in the inhibition of angiogenesis. Here we also report a new mechanism where the Mdm2 oncoprotein can indirectly inactive p53 through the regulation of the tumor suppressor VHL. In response to hypoxia, VHL can bind p53, which results in activation of several anti-angiogenic targets of p53 such as THBS1 and COL18A1. Mdm2 regulates the VHL-p53 interaction by conjugating nedd8 to VHL within a region that is important for the VHL-p53 interaction, blocking the induction of anti-angiogenic genes and resulting in a proangiogenic phenotype. Due to its positive regulation of major proangiogenic proteins and its negative regulation of potent inhibitors of angiogenesis, we propose that the oncoprotein Mdm2 is the angiogenic switch. These findings refine our understanding of p53 interactions and activation, specifically for p53-p73 induced cell death and p53-VHL inhibition of angiogenesis. / 2020-08-05

La expresión de hemoxigenasa 1 (HO-1) ha demostrado estar sobre-regulada en el cáncer colorrectal (CCR), pero el papel que desempeña en este tipo de cáncer aún no se ha dilucidado. Los objetivos de este estudio han sido analizar la expresión de HO-1 en CCR invasivo humano, evaluar su correlación con parámetros clínicos -histopatológicos e investigar los mecanismos por los cuales la enzima influye en la progresión tumoral. Se confirmó que HO-1 fue sobre-expresada en CCR invasivo humano y se encontró que la expresión de la enzima se asocia con un mayor tiempo de supervivencia global de los pacientes. Además, se observó, en un modelo de CCR en ratas inducido químicamente con 1,2-dimetilhidrazina que la expresión total y nuclear de HO-1 aumenta con la progresión tumoral. El estudio de los mecanismos implicados en la acción de la HO-1 en CCR demostró que la proteína reduce la viabilidad celular a través de la inducción de la detención del ciclo celular y de la apoptosis; y que se requiere la proteína supresora de tumores p53 funcional para estos efectos. Esta reducción en la viabilidad celular se acompaña por la modulación de los niveles de p21, p27, ciclina D1, Bax, uPARP y por la modulación de las vías de caspasa-3, Akt y PKC´s. También, en modelos animales murinos, se demostró que la modulación genética y farmacológica de HO-1 reduce la tasa de crecimiento y la carga tumoral cuando p53 se encuentra en su estado salvaje. En adición, se observó que la sobre-expresión de HO-1 reduce la migración e invasión celular. En conclusión, estos resultados demuestran un papel antitumoral de HO-1 apuntando a la importancia de la condición de p53 en esta actividad antitumoral. / The expression of heme oxygenase-1 (HO-1) was shown to be up-regulated in colorectal cancer (CRC), but the its role in this type of cancer has not yet been elucidated. The objectives of this study were to analyze the expression of HO-1 in human invasive CRC, to evaluate its correlation with clinical-histopathological parameters and to investigate the mechanisms by which the enzyme affects tumor progression. It was confirmed that HO-1 was over-expressed in invasive human CRC and was found that the expression of the enzyme is associated with an increased overall survival time of patients. Furthermore, it was observed in a chemical animal model of CRC induced in rats with 1,2-dimethylhydrazine that the total and the nuclear expression of HO-1 increased with tumor progression. The study of the mechanisms involved in the action of HO-1 protein in CRC, showed that the protein reduces cell viability through induction of cell cycle arrest and apoptosis; and functional suppressor protein p53 is required for this purpose. This reduction in cell viability is accompanied by modulation of the levels of p21, p27, cyclin D1, Bax, uPARP and modulation of caspase-3, Akt and PKC's pathways. Also, in animal murine models, it was demonstrated that genetic and pharmacological modulation of HO-1 reduces the rate of growth of tumors and the tumor load when wild type p53 is present. In addition, we observed that overexpression of HO-1 decreases migration and cell invasion. Altogether, this results show an antitumor role of HO-1 pointing to the importance of p53 status with its antitumor activiy.

Low-grade diffuse astrocytomas have an intrinsic tendency for malignant progression but the factors determining the kinetics of this process are still poorly understood. We report here the case of a male patient who developed a fibrillary astrocytoma at the age of 33 years and who underwent six surgical interventions over a period of 17 years without radiotherapy or chemotherapy. The first three biopsies spanned a period of 11 years and led to the diagnosis of low-grade, diffuse astrocytoma (WHO grade II), with a growth fraction (MIB-1 labeling index) of 2.3-3.7%. The fourth to sixth biopsies showed histological features of anaplastic astrocytoma (WHO grade III), with growth fractions between 5.0 and 10.5%. The fraction of gemistocytic neoplastic astrocytes also increased, from 0.3% in the first biopsy to 17.5% in the last biopsy and preceded the increase in proliferative activity and transition to anaplastic astrocytoma. The fraction of tumor cells immunoreactive to BCL-2 increased from 0.3% to 8.2%. A p53 mutation in codon 273 (CGT→TGT, Arg→Cys) was identified in the first biopsy and persisted throughout the course of the disease. However, the fraction of cells with p53 protein accumulation increased significantly during progression, from 3.2% in the first biopsy to 13.7% in the last. The absence of additional genetic alterations (PTEN mutations, loss of chromosome 10 and 19q) may be responsible for the slow progression and lack of glioblastoma features even after a 17-year disease duration.

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