臺灣博碩士論文加值系統

靈芝蛋白（LZ-8）是一種由Ganoderma lucidum中所萃取出的蛋白質，先前研究發現LZ-8可以調節免疫系統以及抑制癌細胞的生長，本篇研究主要著重在rLZ-8對於人類非小細胞肺癌之影響以及其中的分子機轉。人類非小細胞肺癌當中，有大於百分之六十的腫瘤有過度表現表皮生長因子受器的現象，而過度表現表皮生長因子受器大多造成較差的預後，在表皮生長因子受器高度活化的情況下，會促使癌細胞的增生、分化、轉移以及增加侵襲性等。表皮生長因子受器在接上配體（ligand）後，會促使表皮生長因子受器泛素化（ubiquitination），並且內吞到細胞當中分解。在本篇研究，我們發現rLZ-8可以抑制人類非小細胞肺癌的增生，並且降低表皮生長因子受器的表現。我們發現rLZ-8調控表皮生長因子受器的表現主要是經由Casitas B-lineage Lymphoma（Cbl）此種E3泛素連接酶，增加表皮生長因子受器的泛素化，並且促使表皮生長因子受器走向由網格蛋白（clathrin）調控的內吞作用所分解。總結上述，rLZ-8增加表皮生長因子受器受到Cbl的泛素化，進而使表皮生長因子受器走向clathrin調控的內吞作用，促使表皮生長因子受器分解，進而抑制人類非小細胞肺癌的增生。本篇的研究結果顯示，rLZ-8未來可開發成為抗人類非小細胞肺癌增生的新分子藥物或是做為輔助人類非小細胞肺癌治療的新藥物。

Ling Zhi-8 (LZ-8), a immune-modulatory protein that has been isolated from Ganoderma lucidum. LZ-8 is related with enhancing human immune system and inhibiting cancer cells proliferation and metastasis. In this study, we investigate the molecular mechanisms of recombinant LZ-8 (rLZ-8) in non-small-cell lung cancer (NSCLC). The overexpression of epidermal growth factor receptor (EGFR) has been observed in more than 60% of metastatic NSCLC tumors and correlated with poor prognosis. EGFR-mediated signal transductions play an important role in cell proliferation and tumor progression. Upon the stimulation of EGF, EGFR is internalized through both clathrin-mediated endocytosis and non-clathrin endocytosis, and degraded by the ubiquitin-proteasome/lysosome pathways. Our data indicated that rLZ-8 down-regulates EGFR expression in a time-dependent manner. We also demonstrated that rLZ-8 down-regulates EGFR by enhancing Cbl-dependent degradation of EGFR. We found that depletion of c-Cbl and Cbl-b restored EGFR expression. Furthermore, rLZ-8 down-regulates EGFR by promoting EGFR internalized through the clathrin-mediated endocytosis. This study suggests that rLZ-8 may be a useful therapy in treatment of NSCLC that express EGFR.

Abstract i
中文摘要 ii
Introduction 1
Materials & methods 4
Cell lines 4
Preparation of recombinant LingZhi-8 (rLZ-8) protein 4
Reagents and antibodies 4
MTT assay 5
Western blot analysis 5
Plasmid constructs and cell transfection 5
Transfection of shRNAs 6
Statistical analysis 7
Result 8
rLZ-8 inhibits proliferation of EGFR-expressing NSCLC cells 8
rLZ-8 inhibits NSCLC cells proliferation via EGFR 8
rLZ-8 suppresses EGFR protein expression in EGFR-expressing NSCLC cells 9
The Cbl ubiquitin ligase plays an important role in rLZ-8 suppresses EGFR expression in NSCLC cells 9
Depletion of clathrin heavy chain recovers rLZ-8-induced down-regulation of EGFR in EGFR-expressing NSCLC cells 10
Combinations of gefitinib or cisplatin with rLZ-8 synergistically inhibit the growth of NSCLC cells 11
Discussion 13
Reference 16
Figure legend 23
Figure 1. rLZ-8 inhibits proliferation of EGFR-expressing NSCLC cells. 23
Figure 2. To further confirm the involvement of EGFR in NSCLC cells growth. 25
Figure 3. To determine that rLZ-8 inhibits NSCLC cells proliferation via EGFR. 27
Figure 4. rLZ-8 down-regulated EGFR protein expression in EGFR-expressing NSCLC cells. 30
Figure 5. Cbl tyrosine Y731 phosphorylation was increased in NSCLC cells after treated with rLZ-8. 32
Figure 6. EGFR-expressing NSCLC cells were transfected with Cbl targeted shRNA. 34
Figure 7. Knockdown Cbl recovers rLZ-8-induced down-regulation of EGFR in EGFR-expressing NSCLC cells. 38
Figure 8. EGFR-expressing NSCLC cells were transfected with Clathrin targeted shRNA. 42
Figure 9. rLZ-8 does not promote EGFR degradation under knockdown of clathrin heavy chain. 46
Figure 10. Co-treatment with the rLZ-8 greatly enhances the cytotoxicity of gefitinib and cisplatin in NSCLC cells. 50
Appendixes 53
Table I. Sequence of shRNA 53

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