線粒體 Lon 通過調節口腔癌中的鈣信號來驅動順鉑耐藥

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姓名	譚麗雅(Vidhya Tangeda) 查詢紙本館藏	畢業系所	生命科學系
論文名稱	線粒體 Lon 通過調節口腔癌中的鈣信號來驅動順鉑耐藥 (Mitochondrial Lon drives cisplatin resistance by regulating calcium signaling in oral cancer)
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摘要(中)	Cisplatin is the first-line chemotherapy drug for oral cancer treatment, but patients have always been a problem to be solved for patients with recurrence and drug resistance after cisplatin treatment. Since cisplatin kills cancer cells because it produces oxidative radicals, which in turn damage DNA and causes cell death, and because mitochondria are the most important source of oxidative radicals produced within cells, we explore in this paper how the mitochondrial pressure protein Lon affects the mechanism of action of cisplatin resistance by regulating oxidative stress. The findings of this paper show that cisplatin treatment causes addition modifications to mitochondrial DNA formation, which leads to mitochondrial incapacitation and induces oxidative stress. It was found that cisplatin-induced oxidative pressure increased the performance of Lon protein in oral cancer cells, while increased Lon led to the production of cisplatin resistance; conversely, the decreased content of Lon made cancer cells sensitive to cisplatin. Next, an increase in cisplatin-induced oxidative pressure was observed, resulting in a decrease in the concentration of calcium ions in the mitochondria, and with an increase in Lon, the granules calcium ions were discharged from the mitochondrium through the Lon activation calcium ion discharge channel NCLX, thereby increasing the concentration of calcium ions in the cytoplasm. As a result, an increase in calcium ions in the cytoplasm activates the tyrosine kinase PYK2, as well as downstream SRC-STAT3 pathways, and activates the anti-apoptotic protein Bcl-2 and the inflammatory cytokine IL-6. Through its companion protein function, Lon binds directly to NCLX and stabilizes NCLX and activates its function. Indeed, the use of the NCLX inhibitor CGP37157 attenuates the Lon-activated calcium ion message pathway and increases the sensitivity of cancer cells to cytotoxicity of cisplatin. Animal experiments have confirmed that high Lon manifestations in cancer cells increase cisplatin resistance, which can be overcome by inhibiting NCLX and reducing mitochondrial calcium concentrations. At the clinical end, protein expression of Lon and NCLX was found to be positively correlated in the expression of the two in tumor tissues of patients with oral cancer. In summary, this paper has found a novel mechanism of resistance to cisplatin by mitochondrial Lon, which affects NCLX activity by binding and regulating mitochondrial calcium ion concentrations to overcome drug resistance caused by cell death. In the future, it is expected that Lon inhibitors can be used to solve the potential use of cisplatin resistance, and it is hoped that they can be applied to oral cancer patients with chemotherapy resistance and obtain better clinical treatment results.
摘要(英)	Cisplatin is a first-line chemotherapeutic drug for oral cancer treatment but the resistance is still a persistent problem to be addressed. As mitochondria are the important source of the drug (cisplatin) induced oxidative stress; here, we explored the mechanisms of mitochondrial matrix stress-response protein Lon′s role in cisplatin resistance. Our results showed that cisplatin-formed mtDNA adducts led to mitochondrial dysfunction and induced oxidative stress. Cisplatin-induced oxidative stress upregulated Lon protein expression in oral cancer cells. Upregulation of Lon increased cisplatin resistance; on the contrary, Lon downregulation sensitized cells to cisplatin. Next, we observed that cisplatin-induced mitochondrial dysfunction accumulated excess mitochondrial calcium, intriguingly Lon upregulation effluxes this calcium through activation of NCLX, a major mitochondrial calcium exchanger, and thereby increased intracellular calcium. The increase in cytosol calcium, activated tyrosine kinase PYK2 and its downstream SRC-STAT3 axis to trigger an anti-apoptotic protein Bcl-2 and an inflammatory cytokine IL-6. Lon directly interacted with and stabilized NCLX through its chaperone function. NCLX inhibitor CGP37157 attenuated calcium signaling induced by Lon and sensitized cancer cells to cisplatin cytotoxicity. In vivo studies confirmed that cancer cells with Lon upregulation are resistant to cisplatin and this resistance overcame by inhibition of NCLX. In clinical, the expression of Lon and NCLX is also positively correlated with that observed in oral cancer patient tumors. In summary, this study unveils the novel retrograde signaling activated by mitochondrial Lon towards resistance to cisplatin-induced DNA damage stress, indicating the potential use of Lon Inhibitors for better clinical outcomes in chemo-resistant cancer
關鍵字(中)	★ 線粒體 ★ Lon ★ NCLX ★ 順鉑耐藥 ★ 鈣信號	關鍵字(英)	★ Mitochondria ★ Lon ★ NCLX ★ Cisplatin resistance ★ Calcium signaling
論文目次	Table of contents Abstract …………………………………………………………………………………………i, ii Acknowledgement ……………………………………………………………………................ iii Table of contents ………………………………………………………………………………… iv List of figures …………………………………………………………………………………… vii Abbreviations ………………………………………………………………………………….. viii Chapter I. Introduction …………………………………………………………………................ 1 1.1 Cisplatin mechanism of action ………………………………………………………. 1 1.2 Cisplatin resistant mechanisms ………………………………………......................... 1 1.3 Lon role in tumor ……………………………………………………………………. 2 1.4 Structural and functional aspects of Lon …………………………………...................3 1.5 Calcium role in mitochondria ……………………………………………................... 5 1.6 NCLX structure, function and its regulation by kinases ……………………………...6 1.7 PYK2 role in tumorigenesis …………………………………………………………..8 1.8 Research Objectives …………………………………………………………............. 9 Chapter II. Material and Methods ………………………………………………………………. 10 2.1 Cell Culture details ……………………………………………………………….…. 10 2.2 List of antibodies and reagents ………………………………………………………10 2.3 Plasmids ……………………………………………………………………..............12 2.4 MTS assay …………………………………………………………………………. .12 2.5 Western blot ………………………………………………………………………….13 2.6 Transfection methods………………………………………………………………...13 2.7 Immunofluorescence assay…………………………………………………………. 13 2.8 Calcium assay ……………………………………………………………………….14 2.9 Mitochondrial ROS detection ………………………………………………………..15 2.10 GST pull down assay ……………………………………………………………….16 2.11 In vivo tumor xenograft model ……………………………………………………..16 2.12 Patients and clinical samples procurement………………………………………….17 2.13 Immunohistochemistry assay……………………………………………………….17 2.14 Statistical analysis ………………………………………………………………….17 Chapter III. Results ………………………………………………………………………………18 3.1 Cisplatin damages mtDNA by forming adducts and produce oxidative stress ………18 3.2 Cisplatin-induced mitochondrial dysfunction promotes stress response protein Lon ………………………………………………………………………………………….. 18 3.3 Lon contributes to cisplatin resistance in OSCC cells ……………………………... 19 3.4 Cisplatin-induced mitochondrial dysfunction increased cytosol calcium through Lon mediated activation of NCLX…………………………………………………………... 19 3.5 Lon release excess mitochondrial calcium to cytosol through NCLX under cisplatin treated conditions ………………………………………………………………………..20 3.6 Lon stimulates calcium mediated PYK2-SRC-STAT3 pathway to promote survival of cell after cisplatin treatment…….………………………………………………….......... 21 3.7 Validation of Lon mediated activation of PYK2-SRC-STAT3 pathway……………. 22 3.8 Mitochondria Lon-induced cisplatin resistance is mediated by mitochondria Ca2+- dependent signaling..……………………………………………………………………. 22 3.9 Mitochondrial Lon positively regulates NCLX protein expression…….…………... 23 3.10 NCLX activation is mediated by Lon through direct interaction.………………….. 24 3.11 Lon stabilizes NCLX through its chaperone function……….……………………. .24 3.12 Lon involvement in cisplatin resistance through NCLX is validated In vivo ……... 24 3.13 Clinical evaluation of Lon and NCLX expression in oral tumors ………………….25 Chapter IV. Discussion …………………………………………………………………………. 27 Chapter V. Figures and Legends …………………………………………………………………32 Chapter VI. Conclusion ………………………………………………………………………….53 Chapter VII. Future Prospects ……………………………………………………………………54 Bibliography ……………………………………………………………………………………..55 Achievements ……………………………………………………………………………………62
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指導教授	李岳倫高永旭(Alan Yueh-Luen Lee Yung-Hsi Kao)	審核日期	2022-5-25
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博碩士論文 103881602 詳細資訊