Tato bakalářská práce se zabývá tématem prezentace antigenu a imunitní odpověď při rakovině, konkrétně účinkem metabolitů Disulfiramu.
V teoretické části je stručně popsána rakovina, a to zejména chronická myeloidní leukémie spolu s buněčnými liniemi použitými v experimentech. Jako další je probrána prezentace antigenu a odpověď při rakovině, následovaná imunoterapií. V posledních kapitolách jsme se zaměřili na disulfiram a jeho metabolity a jejich využití nejen při léčbě rakoviny.
Experimentální část pojednává o zvýšení cytotoxické odpovědi, která může být způsobena zvýšením aktivity NK buněk nebo prezentací antigenu zvyšující specifickou cytotoxicitu T lymfocytů. Bylo zjištěno, že ošetření metabolitem disulfiramu zvyšuje cytotoxicitu NK a T buněk proti buněčným liniím K562 a K562 TAX. Jedním z navrhovaných mechanismů je aktivace NF-kB. Inhibice NF-kB by měla zrušit účinek metabolitů disulfiramu při NK a T cytotoxické odpovědi. Pro tento experiment byly použity NF-kB inhibitory: N acetylcystein a aspirin spolu se sulfamethoxazolem jako kontrolou. Dokázali jsme, že inhibice NF-kB pomocí N acetylcysteinu a aspirinu vedla ke zrušení účinku metabolitů disulfiramu na NK a T cytotoxické odpovědi proti buněčným liniím K562 a K562 TAX. Tyto výsledky potvrzují, že aktivace NF-kB by mohla být jedním z mechanismů zodpovědných za účinek disulfiramu proti rakovině.
Anotace v angličtině
The present thesis deals with the antigen presentation and immune response in cancer, namely the effect of Disulfiram metabolites.
In the theoretical part, cancer is briefly described and in particular chronic myeloid leukemia along with the cell lines used in the experiments. Antigen presentation and immune response in cancer followed by immunotherapy are discussed next. In the last chapters, we focused on disulfiram and its metabolites and their use not only in cancer treatment.
The experimental part focuses on the enhancement of cytotoxic response which can be due to an increase in NK cell activity or antigen presentation, increasing specific T lymphocyte cytotoxicity. It was found that a disulfiram metabolite treatment enhances NK and T cell cytotoxicity against the K562 and K562 TAX cell lines. One of the proposed mechanisms is the activation of NF-kB. Inhibition of NF-kB should abolish the effect of disulfiram metabolites on NK and T cytotoxic responses. The NF-kB Inhibitors: N acetylcysteine and aspirin together with sulfamethoxazole as a control were used for this experiment. We showed that the inhibition of NF-kB by N acetylcysteine and aspirin abolished the effect of disulfiram metabolites on NK and T cytotoxic responses against the K562 and K562 TAX cell lines. These results confirm that activation of NF-kB could be one of the mechanisms responsible for disulfiram effect against cancer.
Klíčová slova
rakovina, metabolity disulfiramu, NK cytotoxicita, T cytotoxicita, NF-kB
Klíčová slova v angličtině
cancer, disulfiram metabolites, NK cytotoxicity, T cell cytotoxicity, NF-kB
Rozsah průvodní práce
37 s
Jazyk
AN
Anotace
Tato bakalářská práce se zabývá tématem prezentace antigenu a imunitní odpověď při rakovině, konkrétně účinkem metabolitů Disulfiramu.
V teoretické části je stručně popsána rakovina, a to zejména chronická myeloidní leukémie spolu s buněčnými liniemi použitými v experimentech. Jako další je probrána prezentace antigenu a odpověď při rakovině, následovaná imunoterapií. V posledních kapitolách jsme se zaměřili na disulfiram a jeho metabolity a jejich využití nejen při léčbě rakoviny.
Experimentální část pojednává o zvýšení cytotoxické odpovědi, která může být způsobena zvýšením aktivity NK buněk nebo prezentací antigenu zvyšující specifickou cytotoxicitu T lymfocytů. Bylo zjištěno, že ošetření metabolitem disulfiramu zvyšuje cytotoxicitu NK a T buněk proti buněčným liniím K562 a K562 TAX. Jedním z navrhovaných mechanismů je aktivace NF-kB. Inhibice NF-kB by měla zrušit účinek metabolitů disulfiramu při NK a T cytotoxické odpovědi. Pro tento experiment byly použity NF-kB inhibitory: N acetylcystein a aspirin spolu se sulfamethoxazolem jako kontrolou. Dokázali jsme, že inhibice NF-kB pomocí N acetylcysteinu a aspirinu vedla ke zrušení účinku metabolitů disulfiramu na NK a T cytotoxické odpovědi proti buněčným liniím K562 a K562 TAX. Tyto výsledky potvrzují, že aktivace NF-kB by mohla být jedním z mechanismů zodpovědných za účinek disulfiramu proti rakovině.
Anotace v angličtině
The present thesis deals with the antigen presentation and immune response in cancer, namely the effect of Disulfiram metabolites.
In the theoretical part, cancer is briefly described and in particular chronic myeloid leukemia along with the cell lines used in the experiments. Antigen presentation and immune response in cancer followed by immunotherapy are discussed next. In the last chapters, we focused on disulfiram and its metabolites and their use not only in cancer treatment.
The experimental part focuses on the enhancement of cytotoxic response which can be due to an increase in NK cell activity or antigen presentation, increasing specific T lymphocyte cytotoxicity. It was found that a disulfiram metabolite treatment enhances NK and T cell cytotoxicity against the K562 and K562 TAX cell lines. One of the proposed mechanisms is the activation of NF-kB. Inhibition of NF-kB should abolish the effect of disulfiram metabolites on NK and T cytotoxic responses. The NF-kB Inhibitors: N acetylcysteine and aspirin together with sulfamethoxazole as a control were used for this experiment. We showed that the inhibition of NF-kB by N acetylcysteine and aspirin abolished the effect of disulfiram metabolites on NK and T cytotoxic responses against the K562 and K562 TAX cell lines. These results confirm that activation of NF-kB could be one of the mechanisms responsible for disulfiram effect against cancer.
Klíčová slova
rakovina, metabolity disulfiramu, NK cytotoxicita, T cytotoxicita, NF-kB
Klíčová slova v angličtině
cancer, disulfiram metabolites, NK cytotoxicity, T cell cytotoxicity, NF-kB
Zásady pro vypracování
The enhancement of cytotoxic response can be due to an increase in NK cell activity or antigen presentation increasing specific T lymphocyte cytotoxicity. In order to assess this effect, mononuclear cells from human blood normal donors (PBMC) will be purified by the standard ficoll-hypaque gradient. The cells will then be washed and adjusted at 2 million cells per ml. The cells will be treated with increasing concentrations of disulfiram metabolites for 24 hrs and then the cells will be washed with PBS and challenged against K562 cells to assess NK cytotoxic activity as described previously. Additionally, the PBMC cells of the same donor will be incubated with fixed K562 cells (K562 cells with ethanol) for 7 days. The aim is to generate T cytotoxic cells against K562 cells and test the cytotoxic response in the presence of disulfiram metabolites. The cytotoxic response will be analyzed by the use of propidium iodide using a fluorimeter. Analysis of the results: the results will be analyzed in terms of the efficiency of NK and cytotoxic T cells to kill K562 cells in the treated as compared to the non treated counterpart for every donor. A minimum of 10 normal donors will be used to ascertain the effect of the treatment. One of the proposed mechanisms is the activation of NFkB. Inhibition of NFkB should abolish the effect of disulfiram metabolites on NK and T cytotoxic responses. The NFkB Inhibition will be used to assess the effect of disulfiram metabolites on NFkB cytotoxic responses.
Zásady pro vypracování
The enhancement of cytotoxic response can be due to an increase in NK cell activity or antigen presentation increasing specific T lymphocyte cytotoxicity. In order to assess this effect, mononuclear cells from human blood normal donors (PBMC) will be purified by the standard ficoll-hypaque gradient. The cells will then be washed and adjusted at 2 million cells per ml. The cells will be treated with increasing concentrations of disulfiram metabolites for 24 hrs and then the cells will be washed with PBS and challenged against K562 cells to assess NK cytotoxic activity as described previously. Additionally, the PBMC cells of the same donor will be incubated with fixed K562 cells (K562 cells with ethanol) for 7 days. The aim is to generate T cytotoxic cells against K562 cells and test the cytotoxic response in the presence of disulfiram metabolites. The cytotoxic response will be analyzed by the use of propidium iodide using a fluorimeter. Analysis of the results: the results will be analyzed in terms of the efficiency of NK and cytotoxic T cells to kill K562 cells in the treated as compared to the non treated counterpart for every donor. A minimum of 10 normal donors will be used to ascertain the effect of the treatment. One of the proposed mechanisms is the activation of NFkB. Inhibition of NFkB should abolish the effect of disulfiram metabolites on NK and T cytotoxic responses. The NFkB Inhibition will be used to assess the effect of disulfiram metabolites on NFkB cytotoxic responses.
Seznam doporučené literatury
1. Abas A Lichtman, AH, Pillai S (ed). Cellular and Molecular Immunology. 9th edition 2017. Elsevier. 2. Truesdell J, Miller VA, Fabrizio D. Approach to evaluating tumor mutational burden in routine clinical practice. Transl Lung Cancer Res. 2018 Dec;7(6):678-681. 3. O'Donnell JS, Teng MWL, Smyth MJ. Cancer immunoediting and resistance to T cell-based immunotherapy. Nat Rev Clin Oncol. 2019 Mar;16(3):151-167. 4. Cerezo-Wallis D, Soengas MS. Understanding Tumor-Antigen Presentation in the New Era of Cancer Immunotherapy. Curr Pharm Des. 2016;22(41):6234-6250. 5. Cerezo D, Pe?a MJ, Mijares M, Martínez G, Blanca I, De Sanctis JB. Peptide vaccines for cancer therapy. Recent Pat Inflamm Allergy Drug Discov. 2015;9(1):38-45. 6. Mebarki M, Bennaceur A, Bonhomme-Faivre L. Human-cell-derived organoids as a new ex vivo model for drug assays in oncology. Drug Discov Today. 2018 Apr;23(4):857-863. 7. Heyman B, Yang Y. Chimeric Antigen Receptor T Cell Therapy for Solid Tumors: Current Status, Obstacles and Future Strategies. Cancers (Basel). 2019 Feb 6;11(2). pii: E191 8. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, Iyer AK. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Front Pharmacol. 2017 Aug 23;8:561. 9. Yang EJ, Wu C, Liu Y, Lv J, Sup Shim J. Revisiting Non-Cancer Drugs for Cancer Therapy. Curr Top Med Chem. 2016;16(19):2144-55. 10. Jiao Y, Hannafon BN, Ding WQ. Disulfiram's Anticancer Activity: Evidence and Mechanisms. Anticancer Agents Med Chem. 2016;16(11):1378-1384.
Seznam doporučené literatury
1. Abas A Lichtman, AH, Pillai S (ed). Cellular and Molecular Immunology. 9th edition 2017. Elsevier. 2. Truesdell J, Miller VA, Fabrizio D. Approach to evaluating tumor mutational burden in routine clinical practice. Transl Lung Cancer Res. 2018 Dec;7(6):678-681. 3. O'Donnell JS, Teng MWL, Smyth MJ. Cancer immunoediting and resistance to T cell-based immunotherapy. Nat Rev Clin Oncol. 2019 Mar;16(3):151-167. 4. Cerezo-Wallis D, Soengas MS. Understanding Tumor-Antigen Presentation in the New Era of Cancer Immunotherapy. Curr Pharm Des. 2016;22(41):6234-6250. 5. Cerezo D, Pe?a MJ, Mijares M, Martínez G, Blanca I, De Sanctis JB. Peptide vaccines for cancer therapy. Recent Pat Inflamm Allergy Drug Discov. 2015;9(1):38-45. 6. Mebarki M, Bennaceur A, Bonhomme-Faivre L. Human-cell-derived organoids as a new ex vivo model for drug assays in oncology. Drug Discov Today. 2018 Apr;23(4):857-863. 7. Heyman B, Yang Y. Chimeric Antigen Receptor T Cell Therapy for Solid Tumors: Current Status, Obstacles and Future Strategies. Cancers (Basel). 2019 Feb 6;11(2). pii: E191 8. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, Iyer AK. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Front Pharmacol. 2017 Aug 23;8:561. 9. Yang EJ, Wu C, Liu Y, Lv J, Sup Shim J. Revisiting Non-Cancer Drugs for Cancer Therapy. Curr Top Med Chem. 2016;16(19):2144-55. 10. Jiao Y, Hannafon BN, Ding WQ. Disulfiram's Anticancer Activity: Evidence and Mechanisms. Anticancer Agents Med Chem. 2016;16(11):1378-1384.
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Plný text práce
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Hodnocení vedoucího
Záznam průběhu obhajoby
Obhajobu bakalářské práce studentky Evy Komínkové zahájil prof. RNDr. Milan Navrátil, CSc. Studentka seznámila komisi s hlavními částmi a výsledky své práce. Následovalo přečtení posudku vedoucího práce prof. Juan Bautista De Sanctis, Ph.D. a oponenta Viswanath Das, Ph.D. MSc.
Studentka pak reagovala na připomínky a dotazy.
Prezentace studentky obsahovala tato témata:
- Aims of the thesis
- Cancer - characterisation
- Antigen presentation and immune response in cancer
- Disulfuram and its metabolite CuET
- Working procedure, design of expeirmentT cell cytotoxicity assays
- NK cytotoxicity assays
- Effect of NFkB inhibitors
V rozpravě byly uvedeny následující připomínky či nastoleny tyto problémy:
- Lack of statistical analysis in Graphs 5-8, significancy of effect od NFkB inhibitors
- Mechanism of anticancer effect of CuET
- Effect of disulfuram to proteasome
- Difference between used cell lines
- Effect of disulfuram and possibility to modifie cell response after virus infection
Studentka na dotazy položené oponentem práce a členy komise reagovala adekvátně, se znalostí problematiky.