臺灣博碩士論文加值系統

肝細胞癌（Hepatocellular carcinoma, HCC）是全球第六大流行的癌症類型，也是第三大致命癌症。由於早期肝癌沒有症狀的原因，許多肝癌患者直到晚期階段才被診斷，而此時患者會接受多重酪胺酸激酶抑制劑如Sorafenib的標準治療。然而，僅有少部分病患對Sorafenib有治療反應，且許多患者在接受治療後6個月內就會出現Sorafenib抗藥性。另外，約六成的晚期及末期肝癌的病患會表現惡病質，其臨床表現包括骨骼肌肉萎縮和體重減輕，並會降低Sorafenib治療效果與耐受性，進而降低患者存活。從先前文獻中已知發炎細胞因子在癌症的惡病質中扮演重要角色。在本論文中，我們認為Sorafenib抗藥性的肝癌細胞會分泌更多發炎因子，進而促進肝癌惡病質。目前為止，我們分析我們實驗室建立的Sorafenib抗藥肝癌細胞株，發現在一些Sorafenib抗藥的肝癌細胞株中，會有較高的發炎因子表現，而這些抗藥性細胞株所收集的條件培養液會造成小鼠C2C12肌小管的萎縮。在動物實驗中，我們也發現帶有抗藥性細胞腫瘤的小鼠其肌肉纖維有顯著萎縮。此研究反應出Sorafenib抗藥性的產生可能會惡化晚期肝癌的惡病質表現。

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer type, and the third deadliest cancer in the world. As liver is a silent organ, many HCC patients are not diagnosed until having reached advanced stage, when multi-targeted tyrosine kinase inhibitors such as Sorafenib remain the standard of care. However, only a small portion of patients respond to Sorafenib, and many patients develop Sorafenib resistance within 6 months. Associated with advanced and terminal stage HCC is the clinical syndrome of cachexia, which occurs in roughly 60% of HCC patients. The central hallmarks of cachexia include skeletal muscle depletion (sarcopenia) and weight loss, which associate with decreased response and tolerance to Sorafenib and decreased patient survival. As inflammatory cytokines play an important role mediating cancer-associated cachexia, we hypothesize that Sorafenib-resistant cells secrete higher level inflammatory cytokine, thereby promoting HCC-associated cachexia. Targeting these cytokine-mediated pathways may be a promising therapeutic option to reverse cachectic phenotype. In this thesis, we have shown that Sorafenib-resistant HCC cells expressed higher level of these cytokines, and conditioned medium collected from resistant cells induced myotube atrophy in vitro. In vivo xenograft model also demonstrated elevated serum level of cytokine, and muscle fiber atrophy was noted in Sorafenib-resistant tumor bearing model. Taken together, this study suggested acquired resistance to Sorafenib contributed to development of cachexia in HCC.

摘要 5
ABSTRACT 6
Chapter 1 Introduction 7
1.1 Hepatocellular carcinoma (HCC) 7
1.2 Cancer-associated cachexia 8
1.3 Inflammatory cytokine and multitargeted tyrosine kinase inhibitor (TKI) 10
1.4 Rationale and specific aims 11
Chapter 2 Materials and methods 13
2.1 Cell culture 13
2.2 Conditioned medium (CM) collection and treatment 14
2.3 RNA isolation and real-time quantitative polymerase chain reaction 14
2.4 Xenograft HCC model 15
Chapter 3 Results 16
3.1 Sorafenib-resistance increased expression of cancer stemness-related genes and inflammatory cytokines in HCC cell line 16
3.2 Differentiated C2C12 myotube showed elongated tube-like morphology and differentiation-associated gene signature 17
3.3 Treatment of C2C12 myoblasts with inflammatory cytokine increase cytokine-associated downstream target gene expression 18
3.4 Conditioned medium of Sorafenib-resistant HCC cell lines promoted myotube atrophy in C2C12 myotube model 18
3.5 Anti-cytokine treatment reversed the elevated MuRF1 gene expression in myotube atrophy model induced by HepG2215_R CM 18
3.6 Sorafenib-naïve and Sorafenib-resistant HepG2215 tumors exhibited different behaviors in subcutaneous xenograft model 19
3.7 Both Sorafenib-naïve and Sorafenib-resistant tumor-bearing mice had muscle atrophy. 20
3.8 Sorafenib-resistant and Sorafenib-naïve tumor bearing mice had different multiple organ effect. 20
Chapter 4 Discussions 22
Chapter 5 Figures and tables 24
Figure 1. Characterization of Sorafenib-resistant and naïve HCC cell lines. 24
Figure 2. Gene expression and cytokine secretion level of Sorafenib-naïve and resistant HCC cell lines. 26
Figure 3. Morphology and gene expression analysis of C2C12 myotubes. 27
Figure 4. Cytokine target gene expression in mouse C2C12 myoblast cells treated with human cytokine 28
Figure 5. Morphology and diameter of C2C12 myotubes after conditioned medium treatment. 29
Figure 6. Effect of anti-cytokine on MuRF1 gene expression. 30
Figure 7. Body weight change, tumor volume, and serum cytokine level in HepG2215 and HepG2215_R subcutaneously injected NOD-SCID mice. 31
Figure 8. Grip strength test and voluntary activity wheel running test 32
Figure 9. Gross and microscopic examination of tibialis anterior muscle of tumor-bearing mice 33
Figure 10. Organ size of gastrocnemius muscle, heart, liver, and spleen 34
Chapter 6 References 35

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