臺灣博碩士論文加值系統

肝細胞癌 (Hepatocellular carcinoma，簡稱HCC) 這個侵襲性癌症所伴隨的預後不良症是導致世界上大量癌症病患死亡的原因。然而目前FDA唯一核准用藥蕾莎瓦(Sorafenib) 只能短暫維持病人2-3個月的壽命，因此找出治療肝癌的用藥是迫切需要的。我們過去也發現中草藥GP以及其部分純化物 HH-F3被證明具有抗肝癌以及肝纖維化的能力，然而GP以及其純化物 HH-F3作用在肝癌的機制仍不明。在本篇，我發現了HH-F3參與轉型生長因子β(TGF-β) 以及AMPK的路徑。結果顯示HH-F3可以調控與腫瘤生成、血管新生以及脂肪生成相關的路徑TGF-β，並導致其相關蛋白包括: pSMAD2/3、p38、MEK以及ID 蛋白家族的下調。此外HH-F3可以通過磷酸化AMPK並調控其下游ACC，SREBP-1C，SREBP2，FASN和PGC-1α來抑制細胞的脂肪生合成。同時我們也發現TGF-β能夠抑制HH-F3降低Huh7及HepG2細胞脂肪堆積的能力。綜合上述，我們推斷HH-F3在肝癌中可以調控TGF-β路徑和AMPK路徑並且導致脂肪生成路徑的下調。

Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor prognosis and most common cause of cancer deaths in the world. However, sorafenib is the only target therapy of HCC approved by the FDA and prolongs patient’s survival for 2-3 months. Therefore, it is urgent to identify new drug candidates for the treatment of HCC. To identify the therapeutic agents, we previously found that one of the herb medicine, GP and it partially purified fraction HH-F3 have anti-HCC and anti-fibrosis effects. However, the mechanism of HH-F3 was not clear yet. In this study, I demonstrated that GP and HH-F3 were involved in TGF-β and AMPK pathway. TGF-β signaling pathway is known to regulate tumor progression, angiogenesis, and lipognesis and ROS production in HCC. HH-F3 could down-regulate TGF-β signaling pathway through p-SMAD2/3, p-P38, p-MEK and ID protein family in Huh7 and HepG2 cell lines. Moreover, HH-F3 could inhibit lipogenesis via regulating AMPK and its downstream targets, including ACC, SREBP-1C, SREBP2 FASN and PGC-1α in Huh7 and HepG2 cell lines. In addition, TGF-β could inhibit HH-F3, reversing its reduction of lipid accumulation in Huh7 and HepG2 cell lines. In conclusion, HH-F3 could inhibit lipogenesis via TGF-β pathway and AMPK pathway, and might be a potential therapeutic drug in HCC.

致謝 I
Content II
Chinese Abstract VI
Abstract VII
Introduction 1
Hepatocellular Carcinoma (HCC) 1
Graptopetalun paraguayense (GP) / HH-F3 2
Transforming growth factor beta (TGF-β) pathway 2
Lipogenesis 3
ConsensusPathDB (CPDB) 4
Connectivity Map (Cmap) 5
L1000 profiling 5
Objective 7
Materials and Methods 8
Cell lines and cell culture 8
Culture medium concentrates 8
Oil red O assay 9
SRB assay 9
Protein extraction and concentration measurement 9
Western blotting analysis 10
Antibody 10
Q-RT-PCR 11
Results 13
HH-F3 regulates energy metabolism and TGF-β pathway gene expression signatures in HCC cell lines. 13
HH-F3 suppresses extracellular TGF-β protein level and increases expression level of TGF-β receptor in HCC cell lines. 14
HH-F3 increases TGF-β receptor and suppresses SMAD-dependent and -independent pathway in HCC cell lines. 14
HH-F3 decreases lipid accumulation and fatty acid synthesis in HCC cell lines. 15
TGF-β increases oleic acid (OA) and palmitic acid (PA)-induced lipid accumulation in HCC cell lines. 16
To search for GP/HH-F3 similar drugs via gene expression signatures and Connectivity Map. 17
GP/HH-F3 similar drugs inhibit cell line growth and reduce lipid accumulation in Huh7 cell line 17
Discussion 19
Reference 25
Figures 32
Figure 1. The schematic illustration of using gene expression signatures reveals HH-F3 modulated pathways. 32
Figure 2. Treatment of HH-F3 suppresses extracellular TGF-β level in HCC cell lines. 34
Figure 3. Treatment of HH-F3 increases the level of TGF-β receptor in HCC cell lines. 36
Figure 4. HH-F3 suppresses SMAD-dependent pathway in HCC cell lines. 38
Figure 5. HH-F3 suppresses SMAD-independent pathway in HCC cell lines. 40
Figure 6. HH-F3 suppresses the expression of ID protein family (ID1, ID2, and ID3) in Huh7 and HepG2. 41
Figure 7. HH-F3 reduces oleic acid (OA) and palmitic acid (PA)-induced lipid accumulation in HCC cell lines. 42
Figure 8. GP and HH-F3 suppress fatty acid synthesis pathway in HCC cell lines. 43
Figure 9. HH-F3 decreases the expression of PGC-1α in HCC cell lines. 44
Figure 10. TGF-β increases oleic acid (OA) and palmitic acid (PA)-induced lipid accumulation and inhibits HH-F3-reduced lipid accumulation in HCC cell lines. 46
Figure 11. Search for GP/HH-F3 similar drugs via expression signatures. 47
Figure 12. GP/HH-F3 similar drugs inhibit Huh7 and HepG2 cell lines growth. 48
Tables 51
Table 1. Affymetrix microarray U133plus_2 and L1000 used in this study. 51
Table 2. L1000 expression signatures in HepG2 cells reveal up-regulated pathways. 52
Table 3. L1000 expression signatures in HepG2 cells reveal down-regulated pathways. 55
Table 4. Affymetrix microarray U133 plus_2 expression signatures in HepG2 cells reveal up-regulated pathways. 56
Table 5. Affymetrix microarray U133 plus_2 expression signatures in Huh7 cells reveal down-regulated pathways. 58
Table 6. Affymetrix microarray U133 plus_2 expression signatures in HepG2 cells reveal up-regulated pathways. 61
Table 7. Affymetrix microarray U133 plus_2 expression signatures in HepG2 cells reveal down-regulated pathways. 65
Table 8. Affymetrix microarray U133 plus_2 expression signatures in Mahlavu cells reveal up-regulated pathways. 70
Table 9. Affymetrix microarray U133 plus_2 expression signatures in Mahlavu cells reveal down-regulated pathways. 75
Table 10. TGF-β pathway genes are regulated under HH-F3 treatment in HCC cell lines. *LM: Landmark genes. INF: Inferred genes 78
Table 11. GP/HH-F3 from different sources used in this study. 79
Table 12. GP/HH-F3 similar drugs predicated by L1000 gene signatures and Affymetrix microarray. 80
Table 13. HH-F3 modulates TGF-β related pathway via different kinetics in Huh7 cells. 81
Table 14. HH-F3 modulates TGF-β related pathway via different kinetics in HepG2 cells. 82
Supplementary figure 1. GP/HH-F3 recovers lipid accumulation induced by oleic acid (OA) and palmitic acid (PA) in Huh7. 83
Supplementary figure 2. TGF-β induces TGF-β signaling pathway in HepG2 cell line. 84
Supplementary figure 3. HH-F3 suppresses fatty acid synthesis pathway in Huh7 cell line. 85
Supplementary figure 4. HH-F3 suppresses PGC-1α and p-SMAD3 in HepG2 cell line. 86

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