細胞介白素8（或稱CXCL8, IL-8）是CXC家族趨化激素之一，它可經由其受體CXCR1/2路徑促進腫瘤發展及刺激癌細胞生長、移動、侵入、上皮細胞間質轉化。結合上述IL-8在腫瘤發展所扮演的重要性，我們發展了細胞介白素8類似物-RP4，希望藉其對於CXCR1/2較高的結合親和力，而去抑制腫瘤的進程。在本研究中會針對臨床上較惡性、藥物發展也發生限制的非小型細胞肺癌及胰腺癌。肺腺癌及胰腺癌雖在臨床上皆有一線標準用藥，如艾瑞莎(Gefitinib)及健擇(Gemcitabine)，但在短暫的有效治療後皆相繼產生抗藥性，近期有許多文獻證實，細胞介白素8的分泌和市售藥物產生抗藥性具相關性。因此，在癌症的進程及發展上，針對細胞介白素8及其受體CXCR1/2被認為是具潛力的治療策略。在本研究中，用細胞介白素8類似物-RP4針對具艾瑞莎抗性之非小型細胞肺癌及具健擇抗性之胰腺癌研究抗癌效力。在初期的結果上，我們發現在懸浮的狀態下，RP4發揮更好的削弱癌細胞特性的效果。接著，在具艾瑞莎抗性之非小型細胞肺癌及具健擇抗性之胰腺癌的兩類細胞株上，我們發現RP4不僅抑制癌細胞的生長、移動、侵入同時也使細胞介白素8及其受體CXCR1/2的基因表現降低。RP4的抑癌效果在動物實驗的生存率及腫瘤大小上也表現得十分顯著。綜合上述結果，我們相信細胞介白素8類似物-RP4可發展為一個具潛力的候選藥物。

IL-8 (CXCL8, interleukin-8), a CXC family chemokine, can stimulate the tumor progression and trigger cancer cell growth, migration, invasion, and Epithelial-Mesenchymal Transition (EMT) via its receptors CXCR1/2 (IL-8R) pathway. As stated above, IL-8 plays a crucial role in tumor progression, so we design an IL-8 analogue-RP4, hoping to inhibit tumor development by its high binding affinity with CXCR1/2. In this study, we focus on non-small cell lung cancer (NSCLC) and pancreatic cancer (PDAC) which is malignant and limited drug application in clinical. Although NSCLC and PDAC have standard first-line medicines, e.g. Gefitinib and Gemcitabine, drug resistance occurs after treating few months. Many literatures had demonstrated that commercial drug resistance in NSCLC and pancreatic cancer PDAC is associated with IL-8 secretion recently. Therefore, targeting IL-8 and IL-8R in cancer progression and development is regarded as a potent therapeutic strategy. Herein, we used the IL-8 analogue, RP4, and investigated the anti-cancer activity in Gefitinib-resistant NSCLC cell lines and Gemcitabine-resistant pancreatic cancer cell lines. In initial results, we find RP4 can attenuate the characteristics of cancer cells much more effective in suspended condition. Furthermore, we found it not only inhibits cells’ growth, migration, and invasion but decreases the IL-8/IL-8R mRNA expression in drug-resistant NSCLC cell lines and pancreatic cell lines. The anti-tumor activity of RP4 also shows marvelous results in survival rate and tumor size in vivo. According to above findings, we believe this IL-8 analogue, RP4, can be developed as a potential drug candidate.

摘要................................................................................................................................ I
Abstract ........................................................................................................................ II
Content ....................................................................................................................... III
Figure list ................................................................................................................... V
Table list ..................................................................................................................... VII
Chapter 1 Introduction................................................................................................ 1
1.1 The role of interleukin 8 (IL-8) in tumor progression .......................................... 1
1.2 Non-small cell lung cancer (NSCLC) .................................................................. 1
1.3 Pancreatic cancer (PDAC) ................................................................................... 3
1.4 Gefitinib (Iressa®) drug resistant .......................................................................... 4
1.5 Gemcitabine (Gemzar®) drug resistant ................................................................ 5
1.6 Design of IL-8 analogue ....................................................................................... 6
Chapter 2 Material and Methods ............................................................................... 8
2.1 Cell lines ............................................................................................................... 8
2.2 Wound-healing assay ......................................................................................... 10
2.3 Intercellular junction and attachment of HUVEC .............................................. 10
2.4 Cell viability assay by MTT ............................................................................... 11
2.5 Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
.................................................................................................................................. 12
2.6 Human IL-8 enzyme-linked immunosorbent assay (ELISA) ............................ 12
2.7 Doubling-time assays of suspended cells ........................................................... 13
2.8 Cell proliferation assay by CCK-8 reagent ........................................................ 13
2.9 Colony formation assay ...................................................................................... 14
2.10 Boyden chamber invasion assay ...................................................................... 14
2.11 Flow cytometry in analysis of cell cycle .......................................................... 15
2.12 Analysis in human lung cancer data sets .......................................................... 15
2.13 Mouse tumor xenograft models ........................................................................ 16
2.14 Statistical analysis ............................................................................................ 17
Chapter 3 Results ....................................................................................................... 18
3.1 RP4 application in lung cancer. .......................................................................... 18
3.1.1 RP4 affects lung cancer cell lines in high IL-8/IL-8R expressed-suspended
condition. .................................................................................................................. 18
3.1.2 HUVEC cell-junction can be protected and recovered from IL-8 by RP4. .... 19
3.1.3 RP4 attenuates lung cancer characteristics in vitro. ........................................ 20
3.1.4 RP4 inhibit anoikis-resistant and anchorage-independent growth by G1 phase
arrest. ........................................................................................................................ 21
3.1.5 Anti-tumor effects of RP4 in vivo LL/2 C57BL/6 mouse model. .................. 22
3.2 RP4 and Gefitinib combined use in Gefitinib-resistant NSCLC cell lines. ....... 23
3.2.1 High expression IL-8/IL-8R of NSCLC are reduced by RP4 combined with
Gefitinib in suspended condition.............................................................................. 23
3.2.2 RP4 reduces Gefitinib-resistant NSCLC cells anchorage - independent growth
by cell cycle G1 phase arrest. ................................................................................... 25
3.2.3 RP4 also reduces Gefitinib-resistant NSCLC cells invasion in vitro. ............. 25
3.2.4 Combined usage of Gefitinib and RP4 inhibits lung tumor growth and prolongs
lifespan in vivo. ........................................................................................................ 26
3.3 RP4 and Gemcitabine combined use in Gemcitabine-resistant PDAC cell lines.
.................................................................................................................................. 26
3.3.1 Gemcitabine-resistant PDAC cell lines show high gene expression of IL-8/IL-
8R, but reduced by RP4. ........................................................................................... 27
3.3.2 RP4 can reduce Gemcitabine-resistant PDAC cells invasion ability in vitro. 27
3.3.3 Combined usage of Gemcitabine and RP4 inhibits pancreatic tumor growth and
reduces death in vivo. ............................................................................................... 28
Chapter 4 Discussion and Conclusion ...................................................................... 29
References ................................................................................................................... 58

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