臺灣博碩士論文加值系統

總摘要:
肺癌為一個具有高度轉移能力的癌症，其早期的診斷相當困難，亦為導致癌症病人死亡的重要原因。癌症轉移過程包含細胞外基質(extracellular matrix, ECM)的降解，癌細胞侵入(invasion)到循環系統的血管或淋巴管，並具有移動(migration)及黏附(adhesion)到遠端器官的能力，最終誘發血管新生(angiogenesis)作用使癌細胞不斷增生(proliferation)。因此開發新的治療方式是非常重要的研究主題。有文獻指出，維生素C合併維生素K3 (CK3)在活體內具有抗癌的作用，然而其作用機制仍不清楚。本論文的第一部分，我們利用皮下異體移植肺癌細胞株(Lewis lung carcinoma)的C57BL/6小鼠做為實驗模式，並以腹腔注射的方式給予高(1000 mg vit C/kg + 10 mg vit K3/kg)、低劑量維生素CK3(100 mg vit C/kg + 1 mg vit K3/kg) 並以順鉑(cisplatin, 6 mg /kg)做為正控制組探討維生素CK3在活體內抑制轉移的作用與機制。結果顯示，維生素CK3及順鉑可顯著抑制腫瘤生長、癌細胞轉移到肺臟的情形，且CK3是具有劑量效應的。順鉑使用時會造成副作用，導致小鼠體重明顯下降，而使用CK3治療的小鼠體重則可回復到與控制組無顯著差異的狀態。分析其抗癌機轉，維生素CK3可有效1、降低小鼠血漿中金屬基質蛋白酶(metalloproteinase )-2, 9以及尿素激酶型胞漿素原活化子(urokinase plasminogen activator)；2、增加小鼠肺臟組織中金屬基質蛋白酶抑制劑(tissue inhibitor of metalloproteinase-1)-1, 2、抗轉移蛋白23(non-metastatic protein 23 homolog 1)以及尿素激酶型胞漿素原活化子抑制劑(plasminogen activator inhibitor-1)蛋白質之表現；3、抑制小鼠肺臟組織中金屬基質蛋白酶-2,9蛋白質表現以及4、減少小鼠肺臟組織中增生細胞核抗原(proliferating cell nuclear antigen)表現達到抗轉移作用。
有文獻指出在人體及動物模式中，鉑複合物已被證實可有效抑制腫瘤的生長。在這些鉑複合物中，順鉑是第一個被製造使用的，但卻會造成強烈的腎毒性副作用。而維生素C是一種水溶性維生素及抗氧化劑，有文獻指出維生素C可以保護細胞膜完整及保護DNA免受自由基的攻擊以避免產生心血管疾病和癌症等慢性疾病產生。但目前在活體內並不清楚維生素C合併順鉑使用是否有加乘的抗癌作用且可減輕順鉑導致的腎毒性副作用與氧化傷害。因此在第二部分中我們利用皮下異體移植肺癌細胞株的小鼠做為實驗模式，並以腹腔注射的方式給予高(1000 mg/kg)、低劑量(100 mg/kg)維生素C單獨或合併順鉑(5 mg/kg)探討維生素C合併順鉑在活體內降低順鉑造成的腎毒性及氧化傷害。結果顯示維生素C及順鉑單獨使用皆可顯著抑制腫瘤生長，但維生素C合併順鉑則無加乘抗癌作用。儘管如此，我們發現維生素C合併順鉑使用，可降低小鼠血漿中尿素氮以及肌酸酐的含量，此外，還可顯著的減少肝臟及腎臟中脂質過氧化、蛋白質過氧化以及減少抗氧化酵素GSH的損耗，減少順鉑使用造成的氧化傷害而達到保護作用。
綜合上述，在第一部分我們證實了在活體內，維生素C合併K3不只會抑制原位癌生長，並且具有增加抗癌能力的表現，而此結果之抗癌能力，是與減少腫瘤侵襲與增生能力有關的。此外，在第二部分我們證實了單獨維生素C可抑制小鼠原位癌生長而vit C合併順鉑相加或相乘效應。也證明了維生素C可顯著減少順鉑使用所造成之腎毒性以及氧化傷害之副作用。

Overall abstract:
Lung cancer, a highly metastatic cancers and difficultly diagnosis in an early stage, is one of the most common cause of death for cancer patients death in worldwide. Metastasizing cells must first disseminate from the primary tumor, invade the surrounding tissue, intravasate and extravasate the circulatory system, arrest, initiate angiogenesis and colonize distant site. Therefore, development of novel therapeutic strategy for treatment lung cancer is important. Vitamin C in combination with vitamin K3 (vit CK3) has been shown to inhibit tumor growth and metastasis in vivo, but the mechanism of action is poorly understood. In the first part thesis of, C57BL/6 mice were implanted (s.c.) with Lewis lung carcinoma (LLC) for 9 days before injection (i.p.) with low- (100 mg vit C/kg + 1 mg vit K3/kg) and high- dose (1000 mg vit C/kg + 10 mg vit K3/kg) vit CK3 twice a week for additional 28 days. As expected, vit CK3 or cisplatin (6 mg /kg, as positive control) significantly and dose-dependently inhibited tumor growth and lung metastasis in LLC-bearing mice. Vit CK3 restored the body weight of tumor-bearing mice to the level of tumor-free mice. Vit CK3 significantly decreased activities of plasma metalloproteinase (MMP)-2, -9 and urokinase plasminogen activator (uPA). In lung tissues, Vit CK3 1) increased protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, non-metastatic protein 23 homolog 1 (nm23-H1) and plasminogen activator inhibitor-1 (PAI-1); 2) reduced protein expression of MMP-2 and MMP-9; 3) inhibited the proliferating cell nuclear antigen (PCNA). These results demonstrate that vitamin CK3 inhibits primary tumor growth and exhibits anti-metastastic potential in vivo through attenuated tumor invasion and proliferation.
Platiunm complexes have been shown to inhibit the tumor growth in animals and in human. Among these platinum complexs, cisplatin was the first found to be anti-cancer drugs, but produced strong nephrotoxicity. In the second part of this thesis, we further investigated the role of vit C on nephrotoxicity and oxdative damage caused by cisplatin and the additive effect of vit C in combination with cisplatin. Similarly, we used the same animal model from first part of thesis. Herein, C57BL/6 mice were implanted (s.c.) with Lewis lung carcinoma (LLC) for 9 days before injection (i.p.) with low (200 mg/kg), high-dose (1000 mg/kg) vitamin C in the present or absence of cisplatin (5 mg/kg) twice a week for additional 28 days. Results revealed that vitamin C or cisplatin alone significantly inhibited tumor growth, whereas the inhibitory effect of vitamin C in combination with cisplatin did not exhibit synergistic effect. In addition, we found that vitamin C in combination with cisplatin reduce the nephrotoxicity and oxidative damage caused by cisplatin, as evidenced by decreasing blood urea nitrogen (BUN) and creatinine in plasma and decreasing TBARS, carbonyls and GSH/GSSG ratio in liver and kidney tissues. In summary, we demonstrate that vitamin CK3 inhibits primary tumor growth and exhibits anti-metastastic potential in vivo in first part of thesis, and the results suggest that this effect is related to attenuation of tumor invasion and proliferation. Besides, in second part of this thesis, we demonstrate that vitamin C in combination with cisplatin inhibits primary tumor growth, and reduced the nephrotoxicity and oxidative damage caused by cisplatin in vivo.
chapter 1:
Vitamin C in combination with vitamin K3 (vit CK3) has been shown to inhibit tumor growth and lung metastasis in vivo, but the mechanism of action is poorly understood. Herein , C57BL/6 mice were implanted (s.c.) with Lewis lung carcinoma (LLC) for 9 d before injection (i.p.) with low- (100 mg vit C/kg + 1 mg vit K3/kg), high-dose (1000 mg vit C/kg + 10 mg vit K3/kg) vit CK3 twice a week for additional 28 days. As expected, vit CK3 or cisplatin (6 mg /kg, as positive control) significantly and dose-dependently inhibited tumor growth and lung metastasis in LLC-bearing mice. Vit CK3 restored the body weight of tumor-bearing mice to the level of tumor-free mice. Vit CK3 significantly decreased activities of plasma metalloproteinase (MMP)-2, -9 and urokinase plasminogen activator (uPA). In lung tissues, Vit CK3 1) increased protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1, TIMP-2), non-metastatic protein 23 homolog 1 (nm23-H1) and plasminogen activator inhibitor-1 (PAI-1); 2) reduced protein expression of MMP-2 and MMP-9; 3) inhibited the proliferating cell nuclear antigen (PCNA). These results demonstrate that vitamin CK3 inhibits primary tumor growth and exhibits anti-metastastic potential in vivo through attenuated tumor invasion and proliferation.
chapter 2:
Platiunm complexes have been shown to inhibit the tumor growth in animals and in human. Among these platinum complexs, cisplatin was the first found to be anti-cancer drugs, but produced strong nephrotoxicity. Vitamin C (vit C), an important water soluble vitamin and antioxidant, has been shown to protect cell membranes and DNA integrity against free radicals attack to avoid the progress of chronic disease, such as cardiovascular disease and cancer. However, little is known whether vit C enhanced the anticancer effect and reduces the nephrotoxicity and oxidative damage caused by cisplatin in tumor-xenografted mice. Herein, C57BL/6 mice were implanted (s.c.) with Lewis lung carcinoma (LLC) for 9 days before administration (i.p.) with low (200 mg/kg), high-dose (1000 mg/kg) vit C in the present or absence of cisplatin (5 mg/kg) twice a week for additional 28 days. Results reveal that vit C or cisplatin alone significantly inhibited tumor growth whereas vit C in combination with cisplatin did not exhibit synergistic effect. In addition, vit C ameliorated the nephrotoxicity and oxidative damage caused by cisplatin, as evidenced by decreased blood urea nitrogen (BUN) and creatinine in plasma and decreased TBARS, carbonyls and GSH/GSSG ratios in liver and kidney tissues. In summary, the present study demonstrates that vit C inhibits tumor growth and reduces the nephrotoxicity and oxidative damage in LLC-bearing mice induced by cisplatin.

總摘要 i
Overall abstract iii
Contents v
Tables vi
Figures vii
Abbreviation ix
Background 1
Chapter 1 14
Abstract 15
Introduction 16
Material and method 17
Results 20
Discussion 23
Reference 35
Chapter 2 41
Abstract 42
Introduction 43
Material and method 44
Results 48
Discussion 50
Overall conclusion 54
Reference 67
口試問題與回答 72

List of tables
Chapter 1

Table 1. Effect of vitamin CK3 and cisplatin on body weight and relative organ weight 26
Table 2. Lung metastasis of metastatic tumors in C57BL/6 mice received vitamin CK3 or cisplatin injection for 37 days 27
Chapter 2

Table 1. Effect of vitamin C alone or in combination with cisplatin on body weight and relative organ weight 56

List of Figures
Chapter 1

Fig. 1 Effect of vit CK3 and cisplatin on body weight of C57BL/6 mice 28
Fig. 2 Effect of vit CK3 and cisplatin on primary tumor growth in vivo 29
Fig. 3 Effect of vit CK3 and cisplatin on MMP-2, MMP-9, u-PA activity in the plasma of C57BL/6 mice 30
Fig. 4 Effect of vit CK3 and cisplatin on MMP-2, MMP-9 protein expression in lung tissue of C57BL/6 mice 31
Fig. 5 Effect of vit CK3 and cisplatin on TIMP-1, TIMP-2 protein expression in lung tissue of C57BL/6 mice 32
Fig. 6 Effect of vit CK3 and cisplatin on nm23-H1, PAI-1 protein expression in lung tissue of C57BL/6 mice 33
Fig. 7 Effect of vit CK3 and cisplatin on proliferating cellular nuclear antigen (PCNA) expression in lung tissue of C57BL/6 mice 34

Chapter 2

Fig. 1 Effect of vitamin C alone or cisplatin combination on body weight of C57BL/6 mice 57
Fig. 2A Effect of vitamin C alone or cisplatin combination on BUN levels of C57BL/6 mice 58
Fig. 2B Effect of vitamin C alone or cisplatin combination on creatinine levels of C57BL/6 mice 59
Fig. 3A Effect of vitamin C alone or cisplatin combination on MDA levels in liver tissues of C57BL/6 mice 60
Fig. 3B Effect of vitamin C alone or cisplatin combination on MDA levels in kidney tissues of C57BL/6 mice 61
Fig. 3C Effect of vitamin C alone or cisplatin combination on protein carbonyls levels in liver tissues of C57BL/6 mice 62
Fig. 3D Effect of vitamin C alone or cisplatin combination on protein carbonyls levels in kidney tissues of C57BL/6 mice 63
Fig. 4A Effect of vitamin C alone or cisplatin combination on GSH/GSSG ratio in liver tissues of C57BL/6 mice 64
Fig. 4B Effect of vitamin C alone or cisplatin combination on GSH/GSSG ratio in kidney tissues of C57BL/6 mice 65
Fig. 5 Effect of vitamin C alone or cisplatin combination on tumor volume of C57BL/6 mice 66

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