唾液酸化的過程主要是唾液酸轉移酶(sialyltransferase)催化唾液酸(sialic
acids)鍵結到細胞表面的醣蛋白或醣脂質上醣質部分的末端，過去許多文獻指出唾
液酸化在癌症中常過度表現且伴隨腫瘤細胞的轉移，近年來有文獻指出過度表現
α-2,3-唾液酸轉移酶會促進乳癌細胞的生成，而在本文中我們也證實了在乳癌細
胞株會過度表現α-2,3-唾液酸轉移酶，因此我們推測可以利用唾液酸轉移酶抑制
劑來抑制癌症之轉移。我們實驗室先前與中研院合作的研究利用以石膽酸為基礎
(lithocholic acid-based)的唾液酸轉移酶抑制劑AL10 去測試其在in vitro 和
in vivo 抗轉移的效果，先前的實驗結果顯示在in vivo 中AL10 是相當有效的唾
液酸轉移酶抑制劑並經由抑制β1- integrin 與CXCR4 的唾液酸化來具抗轉移的
效果。乳癌細胞在細胞膜表面會過度表現chemokine receptors，例如：CXCR4 和
CCR7 而使得癌細胞容易轉移到淋巴系統當中，因為淋巴結會高度表現其ligand，
例如：CCL19，CCL21，SDF-1。在本文當中，經我們實驗證明AL10 可以抑制高度
表現α-2,3-唾液酸轉移酶的 MDA-MB231 的侵犯、抗凋亡、增生的能力。而實驗
結果指出AL10 是藉由減少CCR7 唾液酸化並減少下游訊息傳遞分子ERK 及p-38 的
表現進而抑制CCL19 所誘導的侵犯能力以及細胞抗凋亡的能力，此外，AL10 可藉
由減少 CCR7 唾液酸化並減少下游訊息傳遞分子p-38 的表現量，或是經由降低磷
酸化AKT 的表現使得細胞週期的蛋白cyclinD1 降解而抑制細胞的增生。綜合我們
的實驗結果顯示CCR7 受體的唾液酸化對細胞激素CCL19 所引發之乳癌細胞生
長、侵犯與抗失巢凋亡扮演重要的角色。

Sialylation is catalyzed by sialyltransferases (STs) that adding sialic acids to the
terminal positions of oligosaccharide of glycoproteins and glycolipids. This process is
frequently enhanced in cancer and is associated with increased cancer metastasis. Recent
studies demonstrated that over-expression of ST3Gal-I promotes mammary
tumorigenesis. In our experiments, we also find overexpression of α-2,3-ST in breast
cancer cells. We previously synthesized a lithocholic acid-based ST inhibitor AL10 and
demonstrated its anti-metastatic effect in vitro and in vivo. Our results showed that
AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo
via suppression of sialylation of beta1 integrin and CXCR4. Breast cancer cells
expressing high level of chemokine receptors CXCR4 and CCR7 are prone to exhibit
lymphatic metastasis because their cognate ligands CCL19, CCL21 and SDF-1 are
continuously expressed by lymphatic endothelial cells. In this study, we demonstrate
that AL10 can inhibit invasion, proliferation and induce anoikis of
α-2,3-ST-overexpressing MDA-MB231 human breast cancer cells. Our results indicate
that inhibition of CCL19-induced invasion and CCL19-reduced anoikis by AL10 are
associated with reduced sialylation of CCR7 and attenuated activation of the
downstream signaling mediator ERK and p38. In addition, AL10 can inhibit
proliferation by reducing activation of AKT via CCR7 sialylation independent pathway
and p-38 via CCR7 sialylation dependent pathway which results in ubiquitin-dependent
cyclin D1 degradation. Taken together, we conclude that sialylation of CCR7 is critical
for CCL19-stimulated proliferation, invasion and anti-anoikis in breast cancer cells.

Contents
Abstract (in Chinese) …………………………………………… iii
Abstract (in English) …………………………………………… iv
Contents …………………………………………… v-vi
Abbreviations …………………………………………… vii
Introduction …………………………………………… 1-4
Specific aim …………………………………………… 5
Materials and Methods …………………………………………… 6-11
1. Cell lines
2. Primers
3. Antibodies
4. Drugs
5. Cell culture
6. MTT assay
7. Extraction of RNA purification
8. Two-step RT-PCR(Reverse—transcription polymerase chain reaction)
9. Lectin affinity precipitation
10. Immunoprecipitation
11. Western blot analysis
12. In vitro invasion assay
Results ……………………………………………… 12-17
Discussion ……………………………………………… 18-20
Figures ……………………………………………… 21-35
References ……………………………………………… 36-43
Appendix ……………………………………………… 44-48

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