Trimetilacija lizina 27 u histonu H3 (H3K27me3) u odgovoru stanica raka na cisplatinu u uvjetima in vitro

Tomljanović, Marko (2023) Trimetilacija lizina 27 u histonu H3 (H3K27me3) u odgovoru stanica raka na cisplatinu u uvjetima in vitro. Doctoral thesis, Rudjer Bošković Institute.

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Abstract

Cisplatin is used to treat a number of various malignant tumors. The effectiveness of the treatment is limited by the occurrence of serious side effects and/or the cancer cells develop resistance to the treatment. Over the last 18 years, epigenome modulating compounds have been used to treat cancer. Epigenetic changes are involved, at least in part, in the development of cancer cell resistance to a number of antitumor drugs. An important epigenetic mark which is involved in development of resistance is trimethylation of lysine at the position 27 in histone H3 (H3K27me3). This repressive mark is established by the enzyme EZH2, which can be selectively inhibited by the drug tazemetostat. The removal of this mark is catalyzed by the enzymes KDM6A and KDM6B, which can be inhibited by the compound GSK-J4. The aim of this work was exploration of the effect of tazemetostat and GSK-J4, on sensitivity of cancer cells to cisplatin. The research was conducted using cells originating from head and neck (Detroit 562 and FaDu) and colon malignant tumors (HT-29). Combinations of tazemetostat and GSK-J4 with cisplatin have not been sufficiently, if at all, investigated in these models. Additionally, the effect of these compounds in relation to the content of glucose in the nutrient medium was investigated. The cellular response to cisplatin was cell-type specific and was measured with functional cellular assays (viability assay, proliferation assay). FaDu cell line proved to be the most sensitive to cisplatin, Detroit 562 was moderately sensitive, while HT-29 cells were the most resistant. Combining cisplatin with tazemetostat did not increase the effect of cisplatin with respect to the rate of FaDu and Detroit 562 proliferation, while in colon cancer cells, HT-29, tazemetostat antagonized the effect of cisplatin. Contrary to tazemetostat, the compound GSKJ4 exerted synergistic effect with cisplatin, which was obvious through the reduced proliferation rate of Detroit 562 and HT-29 cells. This effect was absent in FaDu cell lines. The effects of combined treatments did not differ significantly regarding the concentration of glucose in the medium. The underlying causes of the changes observed were investigated at the molecular-genetic level, by measuring the transcriptional activity, as well as the protein level of the selected genes involved in the regulation of the cell cycle and their protein products. Cisplatin application was associated with an increase of CDKN1A (P21) transcription in all cell lines. However, in Detroit 562 and FaDu, it was joined with a decrease of the P21 protein in both the total proteins and in the cytoplasmic protein fraction. This decrease was not observed in HT-29 cells. Tazemetostat caused an increase of P21 in all cell lines. Also, administration of tazemetostat, whether alone or in combination with cisplatin, caused an increase of TGFB1 168 HT-29 cells, on both, transcriptional and protein level. This increase coincides with increased proliferation under these specific experimental conditions. This work will serve as the basis for additional research that will explore the possibility of combining inhibition of enzymes KDM6A and KDM6B with cisplatin application, in preclinical cancer models. This in vitro research showed the potentially dangerous side effects of tazemetostat should it be used in patients with colon cancer.

Item Type:

Thesis (Doctoral thesis)

Uncontrolled Keywords:

proliferation; apoptosis; cisplatin; tazemetostat; GSK-J4; P21; TGF-B1; cyclin D2

Subjects:

NATURAL SCIENCES > Biology
BIOMEDICINE AND HEALTHCARE > Basic Medical Sciences

Divisions:

Division of Molecular Medicine

Projects: