||The study on the role of O6-methylguanine-DNA methyltransferase in therapeutic efficacy of cisplatin and its clinical significance in nasopharyngeal carcinoma
||Institute of Clinical Pharmacy and Pharmaceutical sciences
鼻咽癌(Nasopharyngeal carcinoma)為一發生於鼻咽腔上皮的癌症並好發於台灣。在局部晚期(locally-advanced)的病患，以順鉑(cisplatin)為基礎的同步化電療(concurrent chemoradiotherapy)是目前此類患者的標準治療方式；但五年的存活率大約只有百分之六十。如何克服鼻咽癌細胞對順鉑的抗藥性，將有助於改善鼻咽癌臨床治療之成效。順鉑為白金類的化療藥物並且廣泛使用在目前癌症病患的治療。其主要作用機轉為與脫氧核醣核酸(DNA)結合形成股內交叉鏈結(intrastrand cross-linking)，股間交叉鏈結（interstrand cross-linking），或與蛋白質之間形成交叉鍊結，進而抑制癌細胞複製。O6-甲基鳥糞嘌呤-脫氧核醣核酸-甲基轉化酶 (MGMT) 的主要的功能，為轉移鳥糞嘌呤第六位置氧上之甲基至酵素自身內的胱氨酸(cysteine)殘基上，可用來防護O6-alkylguanine烷化劑的毒性。但此轉化酶是否會影響順鉑在鼻咽癌的療效、作用機轉及臨床意義，目前尚無明確的結果報告。本研究首先使用小干擾核醣核酸技術(small interfering RNA)抑制兩株人類鼻咽癌細胞，HONE-1及TW01細胞中 MGMT蛋白的表現，並進而探討MGMT在癌細胞中的表現與順鉑治療之關聯性。接著，我們會去探討MGMT的表現與癌細胞對於DNA修復能力的影響，並探討在順鉑治療的情況下，MGMT被泛素(ubiquitin)化且降解(degradation)的機制。最後，藉由分析83位接受以順鉑為基礎同步化電療的鼻咽癌患者之病理檢體，分析MGMT表現量對於此類病患預後之影響。首先，我們發現癌細胞中MGMT的表現量越高，順鉑對於癌細胞的細胞毒性就會較差，去氧核醣核酸中白金含量也隨之下降。此外藉由host reactivation assay可以發現MGMT蛋白的表現量增加，也會加強癌細胞對於順鉑產生之DNA傷害的修復能力。更重要的是，我們發現MGMT蛋白可以與順鉑造成傷害之去氧核醣核酸形成鍵結；此外，在順鉑治療之後，MGMT蛋白與泛素有結合之現象，並被蛋白酶體(proteasome)降解。而在83位鼻咽癌病人檢體的研究中，我們也發現若病人MGMT 蛋白表現量越高，其接受以順鉑為基礎之同步化電療後的無病存活期(progression-free survival)及整體存活(overall survival)期越短。多變數分析也指出MGMT蛋白之表現為這群鼻咽癌病患預後之獨立影響因子。綜合上述的研究，我們發現O6-甲基鳥糞嘌呤-脫氧核醣核酸-甲基轉化酶在順鉑於鼻咽癌的療效中扮演重要決定因子。此結果提供了調控此蛋白之表現，來改善鼻咽癌的臨床療效的理論基礎。
Nasopharyngeal carcinoma (NPC) is an endemic head-and neck epithelial malignancy in Taiwan. Currently, Cisplatin (CDDP)-based concurrent chemoradiotherapy (CCRT) has become the standard of care for locally-advanced NPC patients but the 5-year overall survival rate is approximately 60% in these patients. How to overcome the drug resistance of CDDP may provide opportunities to improve clinical outcomes of NPC. CDDP is an important anti-cancer drug commonly used in various human cancers, including NPC. It is generally believed that DNA adducts formation, including DNA-protein cross-links, DNA monoadducts , interstrand and intrastrand DNA cross-links, is the cytotoxic target for CDDP. O6-methylguanine–DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from the O6-position of guanine to an internal cysteine residue; it has been well-characterized to be a therapeutic determinant of O6-alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we aim to find out the regulations and interactions between MGMT and CDDP in NPC cell lines, HONE-1 and TW01, using the RNA interference system. The assays for DNA repair capacity, ubiqutination and degradation of MGMT proteins following CDDP treatment were also examined. The relationship between MGMT expression and clinical survivals in 83 NPC patients receiving CDDP-based concurrent chemoradiotherapy (CCRT) was analyzed. Initially, we showed that MGMT-proficient cells were highly resistance to the cytotoxic effects of CDDP as compared to MGMT-deficient cells. Further studies showed that the formation of platinum-DNA adducts after CDDP exposure was significantly lower in MGMT-proficient cells than in MGMT-deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP-induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP-induced DNA damages. Subsequently, CDDP-bound MGMT protein became ubiquitinated and was degraded through ubiquitin-mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP-based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression-free survival (PFS; P=0.022) and overall survival (OS; P=0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (P=0.01, hazard ratio 2.23) and OS (P=0.018, hazard ratio 2.14). In conclusion, our findings indicate that MGMT not only plays a major role in determining the efficacy of CDDP but also is a potential therapeutic target to improve the clinical outcomes of patients with NPC.
Abstract in Chinese: I
List of the content: VIII
List of tables: XI
List of figures: XII
Chapter One: Introduction: 1
1.1 Introduction of nasopharyngeal carcinoma (NPC): 2
1.2 General treatment and the therapeutic effect of cisplatin (CDDP) in NPC: 2
1.3 The cytotoxic mechanism of CDDP action: 3
1.4 The underlying mechanisms of drug resistance to CDDP: 4
1.5 The DNA repair function of O6-methylguanine–DNA methyltransferase (MGMT): 5
1.6 The correlation MGMT and resistance to O6- alkylguanine alkylating agents: 5
1.7 The prognostic role of MGMT in human cancers: 6
1.8 The correlation between MGMT and CDDP: 7
1.9 The specific aims of this study: 7
Chapter Two: Materials and Methods: 9
2.1 General approaches of each aim in this study: 10
2.2 Materials: 11
2.3 Cell culture: 11
2.4 Silencing of MGMT using siRNA transfection: 12
2.5 Western blot analysis: 12
2.6 Growth inhibition assay: 13
2.7 Cell apoptosis analysis by flow cytometry: 13
2.8 Platinum-DNA adduct measurement by enzyme-linked immunosorbent assay: 13
2.9 Host cell reactivation assay: 14
2.10 DNA affinity precipitation assay: 14
2.11 Immunoprecipitation assay: 15
2.12 Platinum–DNA and platinum-MGMT protein adduct assay by mass spectrometer: 16
2.13 In Vitro ubiquitination assay: 16
2.14 Patients and tumor specimens: 17
2.15 Immunohistochemical staining: 17
2.16 Methylation status of the MGMT CpG island: 18
2.17 Statistical analysis: 18
Chapter Three: Results: 19
3.1 MGMT expression modulates cellular sensitivity to CDDP: 20
3.2 MGMT increases DNA-repair capacity of CDDP-damaged oligonucleotides: 21
3.3 CDDP accelerates MGMT degradation via the ubiquitin-dependent proteasomal pathway: 23
3.4 Expression level of MGMT predicts the survival of NPC patients treated with CDDP-based CCRT: 25
3.5 Expression of MGMT inversely correlates with its methylation status: 25
3.6 Figures and tables: 27
Chapter Four: General Discussion and Future Works: 52
4.1 The initiation and significance of this study: 53
4.2 The potential role of MGMT to modulate CDDP cytotoxicity through the repair to CDDP damaged DNA: 54
4.3 The potential mechanism of MGMT to repair CDDP damaged DNA: 55
4.4 The potential mechanism of the synergistic interaction between CDDP and O6-alkylguanine alkylating agents: 56
4.5 The prognostic role of MGMT expression in NPC: 57
4.6 The methylation status of MGMT promoter in NPC: 57
4.7 Conclusion: 58
4.8 Future works: 59
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