先前的研究文獻表明，IL-8可能會對非小細胞肺癌細胞（NSCLC）賦予EGFR-TKI抗藥性，並且有部分研究也揭示了IL-8及IL-8受體（CXCR1/2）與腫瘤進展、轉移和癌症發展有關。此外，藉由藥物/逆境誘導刺激的IL-8訊號傳遞已被先前文獻證明會賦予癌細胞的化學耐藥性。
在先前的研究中，我們開發了一個高效能CXC-受體拮抗劑，為一IL-8相似物，稱之為pT12S。欲利用此CXC-受體拮抗劑(pT12S)和IL-8共同競爭IL-8受體(CXCR1/2) ，達到阻斷IL-8訊號傳遞鏈對癌症發展的影響。
我們通過RT-qPCR和ELISA檢測IL-8和IL-8受體（CXCR1/2）的表達，用以鑑定具有/不具有艾瑞莎(gefitinib)誘導的HCC827和HCC827GR細胞株之特性。我們的結果展示了pT12S可抑制細胞增殖和細胞存活於艾瑞莎處理誘導之HCC827GR細胞株上。此外，我們設計了一系列實驗，藉由侵襲測定法進一步表明IL-8受體（CXCR1/2）於HCC827GR中的關鍵作用，並測定了pT12S具有抑制細胞侵襲和遷移的能力於艾瑞莎處理誘導之HCC827GR細胞株。
我們的數據表明了pT12S可能發展為一種新型抗癌藥物，能克服晚期肺腺癌患者的EGFR-TKI抗藥性。此項研究突顯了IL-8受體（CXCR1/2）在癌症中所扮演的關鍵角色，並揭示了小分子多肽藥物pT12S作用的功能特性。
此外，pT12S可能有助於EGFR-TKI（艾瑞莎）的聯合治療，以延緩未來臨床治療上NSCLC患者TKI抗藥性的出現。

關鍵字： 介白素-8、CXC-受體 (CXCR1/2)、拮抗劑、抗癌胜肽、EGFR-TKI、EGFR-TKI抗藥性、化療抗藥性、非小細胞肺癌

Previous studies had shown that IL-8 may confer resistance to EGFR-TKI in NSCLC, and several studies had also revealed that IL-8 and IL-8R (CXCR1/2) correlate with tumor progression, metastasis and the development of cancer. Moreover, stress and drug-induced IL-8 signaling had been demonstrated that confer chemoresistance in cancer cells.
In our previous study, we developed a high-performance CXC-receptor antagonist, an IL-8 analogue, called pT12S. Use this CXC-receptor antagonist (pT12S) to compete the IL-8R (CXCR1/2) with IL-8 that blocking the IL-8 signaling pathway in cancer progression and development.
We detected the IL-8 and IL-8R expression to identify the properties of HCC827 and HCC827GR cell lines with/without gefitinib-induced by RT-qPCR and ELISA. Here we showed that pT12S can inhibit cell proliferation and cell survival in HCC827GR with gefitinib-induced stress. Additionally, we designed a serial of experiment to reveal the crucial role of IL-8R in HCC827GR by invasion assay, and examined that pT12S can inhibit cell invasion and migration abilities.
In conclusion, our data suggested that pT12S may develop as a novel anticancer drug and overcome the TKI-resistance for advanced lung cancer patients. This study highlights the crucial role of IL-8R (CXCR1/2) in cancer, and reveals the functional properties of the small-molecular polypeptide drug, pT12S. Furthermore, pT12S may contribute to combination therapy with EGFR-TKI (gefitinib) to prolong the emergence of TKI resistance for NSCLC patients in future clinical treatment.

Keywords: Interleukin-8 (IL-8), CXCR1/2, antagonist, anticancer peptide, EGFR-TKI, EGFR-TKI resistance, chemoresistance, NSCLC

摘要 I
Abstract III
Acknowledgement IV
Content V
Figure List X
Chapter 1 Introduction 1
1.1 Lung cancer 1
1.2 EGFR-TKIs & EGFR-TKI resistance 1
1.3 Alimta® (Pemetrexed disodium) 2
1.4 The ELR+-CXC chemokines and their receptors 3
1.5 The role of interleukin-8 (IL-8/CXCL8) pathway in cancer 4
1.6 Design the antagonist of ELR+-CXC receptors 5
Chapter 2 Material and Methods 7
2.1. Chemical and Reagents 7
2.2. Cell lines 7
2.3. Expression of recombinant IL-8 and G31P-IP10-T12S (pT12S) 7
2.4. Expression and purification of recombinant IL-8 and G31P-IP10-T12S (pT12S) 8
2.5. Enzyme-linked Immunosorbent assay (ELISA) 9
2.6. Real-time quantitative reverse transcription-PCR (RT-qPCR) 9
2.7. Cell cytotoxicity of gefitinib/Alimta by CCK8 10
2.8. Proliferation assay by CCK8 10
2.9. Colony formation 11
2.10. Invasion assay 11
2.11. Wound healing assay 11
2.12. Statistical analysis 12
Chapter 3 Results 13
3.1. Characterization of HCC827/GR cell lines sensitivity or resistance with gefitinib/Alimta 13
3.2. Expression levels of IL-8 and IL-8R (CXCXR1/2) with/without gefitinib-induced in HCC827GR compared with HCC827 13
3.3. pT12S can inhibit cell proliferation and cell survival in HCC827GR with gefitinib-induced stress 15
3.4. The crucial role of CXCR1 and CXCR2 in EGFR TKI-resistance cell line by invasion assay 16
3.5. pT12S effectively inhibited gefitinib-induced cell invasion in HCC827GR 16
3.6. pT12S can attenuate cell migration ability by wound-healing assay 16
Chapter 4 Discussion 18
Chapter 5 Conclusion 21
References 52
Supplementary data 56

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