第一部分：欖仁樹的葉抑制口腔癌細胞其轉移和侵襲之機制探討 第二部分：腫瘤轉移抑制基因其基因多型性與非小細胞肺癌之相關性探討

Part I: Study of the inhibitory effects of Terminalia catappa leaves on the metastasis and invasion of oral cancer cells Part II: Association of polymorphism of the metastasis-suppressor gene with susceptibility to and severity of non-small cell lung cancer

楊嘉欣(Jia-Sin Yang)

指導教授 ： 謝易修 ； 楊順發

中山醫學大學/醫學院/醫學研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00096

摘要

第一部分：
近年來台灣地區十大癌症中口腔癌的發生率和死亡率正在逐年增加中，而癌細胞的轉移往往是臨床治療上最難以根治及導致死亡率增加的主因。欖仁樹的葉是坊間廣為流傳的用藥，具有抗氧化及抗發炎的生物活性。但是到目前為止，對於欖仁樹的葉抑制癌細胞侵襲及轉移作用和其詳細機轉還不是很清楚。據統計約有 90% 口腔癌的患者所罹患的為鱗狀上皮細胞癌，其在口腔的發生部位以舌部最多。因此，我們選用 SCC-4 這株高度惡化的口腔舌癌鱗狀上皮細胞來進行實驗。在本實驗中，利用 50% 酒精萃取的欖仁樹的葉之萃取物處理 SCC-4 細胞株，並利用 MTT assays 分析方法顯示欖仁樹的葉萃取物並不會影響 SCC-4 細胞的存活率，而當癌細胞轉移時常常會伴隨著細胞外基質的分解及細胞移動能力的改變，接著我們利用 modified Boyden chamber assay 發現欖仁樹的葉萃取物具有抑制 SCC-4 細胞的移動與侵襲能力。在 gelatin zymography 與 casein zymography assay 中也發現到欖仁樹的葉萃取物可以抑制 SCC-4 細胞中 MMP-2、MMP-9 及 u-PA 的表現。再利用 western blot 發現欖仁樹的葉萃取物會抑制蛋白酶 MMP-2、MMP-9、u-PA 及其內生性抑制劑 TIMP-1、TIMP-2、PAI-1 的蛋白表現，也會抑制訊息傳遞蛋白 PI3K 與磷酸化的 ERK1/2、JNK1/2、Akt 之表現。而我們更進一步萃取其核蛋白，發現核蛋白中 NF-kB、c-Jun 和 c-Fos 的表現也受到抑制。而 EMSA 的結果也顯示欖仁樹的葉萃取物會抑制 AP-1 及 NF-kB 與 DNA 結合的能力。綜合以上結果，欖仁樹的葉萃取物或許可以應用在預防口腔癌的轉移或輔助口腔癌的治療上。
第二部分：
癌症為台灣地區十大死亡原因之冠，其中又以肺癌為首。而導致台灣肺癌高死亡率的可能原因可歸因於腫瘤的轉移。近年來，腫瘤轉移抑制基因與肺癌的相關性已有許多的研究，而其中又以非轉移基因23 (nm23) 及金屬蛋白酶組織抑制劑 (TIMP) 更為大家所熟知。Nm23 因在高轉移的癌細胞株中表現降低而被命名，而之前也有研究發現人類肺癌細胞在 nm23-H1 基因表現減少時有高度轉移的潛力。而 TIMP 是一個分泌型的蛋白，其主要功能是調節基質金屬蛋白酶 (MMP) 的活性。因此，藉由 MMP 的途徑，TIMP 可以維持結締組織的完整性、抑制癌症形成及腫瘤的轉移與入侵。目前有些研究已經證實 TIMP 基因多型性與許多癌症具有相關性。然而，在非小細胞肺癌中 nm23-H1 其 EcoRI 的基因多型性尚未被研究，而 TIMP-1 其 C372T、TIMP-2 其 C303T 與 G-418C 等位置的基因多型性與台灣非小細胞肺癌的相關性仍未有文獻報導。因此，我們透過 case-control 的實驗設計，收集 255 名非小細胞肺癌患者和其年齡和性別相符且來自健康檢查單位的 303 名健康對照者的 DNA 檢體以聚合酶連鎖反應-限制酶切割片段多型性技術 (PCR-RFLP) 來進行nm23-H1 其 EcoRI 的基因多型性的分析。而在 TIMP-1 及 TIMP-2 的基因多型性分析方面，則收集 343 名非小細胞肺癌患者與 289 名健康對照者，並利用相同的分析方式來探討 TIMP 的基因多型性與非小細胞肺癌的相關性。結果發現，非小細胞肺癌患者和健康對照者間其 nm23-H1 基因的基因型頻率具有顯著差異 (p<0.0001)，再進一步利用邏輯斯迴歸模式發現，在肺癌病人攜帶同型對偶基因型者 (A/A) 相較其他對偶基因型者有較高的危險對比值 (ORs) (OR=4.02; 95% CI=2.39-6.76; p<0.0001)。另外，在不同的腫瘤嚴重程度 (stage) 也有顯著的差異 (p=0.001)。而在 TIMP-1 及 TIMP-2 的分析方面，我們發現在非小細胞肺癌患者與健康對照者中 TIMP-1 C372T 其對偶基因頻率與基因型分佈不具有顯著差異。而 TIMP-2 C303T 與 G-418C 之基因多型性頻率分佈在非小細胞肺癌患者與健康對照者間具有顯著差異。進一步在邏輯斯迴歸模式發現攜帶 TIMP-2 C303T (T/T) 基因型者相較 (C/C) 與 (T/C) 基因型者在肺癌發生有較高的危險對比值 (OR=2.27; 95% CI=1.29-4.02; p=0.005)；而攜帶 TIMP-2 G-418C (C/C) 基因型者相較 (G/G) 與 (G/C) 基因型者在肺癌發生也有較高的危險對比值 (OR=3.22; 95% CI=1.66-6.24; p<0.0001)。研究結果顯示 nm23-H1 基因多型性和非小細胞肺癌其易感受性以及疾病的嚴重程度間有顯著的相關性，而 TIMP-2 C303T 與 G-418C 基因多型性可能與台灣非小細胞肺癌的發生率有密切相關性。

關鍵字

欖仁樹的葉 ； 口腔癌 ； 轉移 ； 腫瘤轉移抑制基因 ； 基因多型性 ； 非小細胞肺癌

並列摘要

Part I:
The incidence and mortality of oral cancer in Taiwan have been constantly increased during the last decade and this increase in mortality could be mainly resulted from the difficulty in treatment caused by metastasis. Terminalia catappa leaves was a popular folk medicine with several proven biological benefits including antioxidant and anti-inflammatory activities. However, the detailed effects and mechanisms of Terminalia catappa leaves on cancer cell invasion and metastasis were still unclear. Since about 90% oral cancers have been identified as squamous epithelium cell carcinoma, particularly in tongue, SCC-4 cells, a malignant oral squamous carcinoma cell line, was used in this study. First of all, SCC-4 cells were subjected to a treatment with 50% ethanol extracts of Terminalia catappa leaves and then MTT assays. Results suggested that Terminalia catappa leaves have no effect on cell viability of SCC-4 cells. The modified Boyden chamber assays revealed that a treatment of Terminalia catappa leaves significantly inhibited the cell motility/invasion capacities of SCC-4 cells. Since that tumor metastasis is accompanied with proteolytic degradation of the extracellular matrix and changed cell motility, MMP-2, MMP-9 and u-PA activity were also studied via gelatin zymography and casein zymography assay to show that they all were inhibited by Terminalia catappa leaves. Furthermore, results from western blot showed that Terminalia catappa leave extract may suppress the protein expression of proteinase MMP-9, MMP-2, u-PA and their proteinase inhibitor TIMP-1, TIMP-2, PAI-1. Terminalia catappa leave extract also inhibited the signal transduction protein expression of PI3K and phosphorylation of ERK1/2, JNK1/2 and Akt while the expression of nuclear protein NF-kB, c-Jun and c-Fos were inhibited as well. The EMSA assay also revealed that the DNA binding activity with AP-1 and NF-kB was also decreased by Terminalia catappa leave extract. In conclusion, Terminalia catappa leaves may be a powerful candidate for a preventive agent against oral cancer development and metastasis.

Part II:
Cancer has been the first leading cause of death in Taiwan and lung cancer has the highest mortality among cancers. Based on extensive studies, metastasis could contribute to the high mortality of lung cancer. In recent years, metastasis-suppressor genes, including nm23 and TIMP, have been intensively studied. For examples, a reduced expression of the metastasis-suppressor gene nm23-H1 was detected in human lung cancer cells with high metastatic potential. Tissue inhibitors of metalloproteinase (TIMP) is a secreted protein involved in the regulation of MMPs activities. Therefore, it was suggested that through the MMP dependent pathway, TIMP could maintain connective tissue integrity, thus inhibit carcinogenesis as well as tumor metastasis and invasion. Several studies have demonstrated association between TIMP gene polymorphisms and various cancers. However, the biallelic EcoRI polymorphism of nm23-H1 gene has not been studied in non-small cell lung cancer (NSCLC). Association of polymorphisms of the TIMP-1 C372T, TIMP-2 C303T and G-418C gene and non-small cell lung cancer has not been reported in Taiwan. Thus, we investigated through a case-control study design, genomic DNA samples of 255 NSCLC patients and 303 controls, which were age and sex-matched and recruited from the health check-up unit, were subjected to polymorphism analysis with polymerase chain reaction-restriction fragment length (PCR-RFLP) technique for nm23-H1 EcoRI polymorphism. To investigate association of polymorphisms of TIMP and NSCLC, genomic DNA samples of 343 NSCLC patients and 289 controls for TIMP-1 and TIMP-2 polymorphism analysis with the same technique. Overall, the genotype frequencies of nm23-H1 gene were significantly different between NSCLC patients and controls (p<0.0001). Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were seen in patients homozygous (A/A) for variant allele (an OR of 4.02, 95% CI 2.39-6.76; p<0.0001). Significant difference was also seen between patients with lung cancers of various stages (p=0.001). However, there was no significant difference in the TIMP-1 C372T allele frequencies or genotype distributions between cases and controls. The distribution of genotype frequencies of TIMP-2 C303T and G-418C were significantly different between lung cancer patients and healthy controls. Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were seen for individuals with TIMP-2 C303T (T/T) genotype against (C/C)/(T/C) genotypes (an OR of 2.27, 95% CI 1.29-4.02, p=0.005), and TIMP-2 G-418C (C/C) genotype against (G/G)/(G/C) genotypes (an OR of 3.22, 95% CI 1.66-6.24, p<0.0001). These results have demonstrated a significant association between the polymorphisms of nm23-H1 gene and the susceptibility to and severity of lung cancer. Furthermore, TIMP-2 C303T and G-418C genetic polymorphisms may be associated with an increased risk of NSCLC in the Taiwan population.

並列關鍵字

Terminalia catappa leaves ； metastasis ； oral cancer ； polymorphism ； metastasis-suppressor gene ； non-small cell lung cancer

參考文獻

廖國盟、陳建仁。肺癌之流行病學特徵、危險因子及防治策略。中華公共衛生雜誌 1997; 16: 375-395.

蔡米山。肺癌的早期診斷。聲洋防癌之聲 2003。

郭壽雄。肺癌。當代醫學 1998; 25: 8-13.

孫安迪。檳榔和癌前期與口腔癌。健康世界 1989; 158: 14-18.

李正喆、郭生興、郭英雄、楊博正、韓俊良。頰黏膜癌-最具代表性的口腔癌。台灣醫學 1997; 1: 638-647.