靈芝蛋白(LZ-8) 是從Ganoderma lucidum 菌絲體所萃取之具有免疫調節功能的蛋白質。我們先前研究顯示LZ-8可以抑制肺癌細胞的增生，降低腫瘤大小；然而目前對於LZ-8對抗癌細胞轉移的機轉尚不清楚。上皮-間質細胞轉換過程目前被認為是癌症轉移最關鍵的指標，且此轉換過程中具有多重訊號傳遞參與調節。上皮生長因子授器調控之訊號傳遞對於腫瘤生長與上皮-間質細胞轉換過程扮演著重要之角色。上皮-間質細胞轉換的樞紐轉錄因子Snail也為上皮生長因子之下游訊息傳遞調節。 本研究探討重組靈芝蛋白rLZ-8抑制上皮生長因子受器調節之肺癌細胞上皮-間質細胞轉換過程。結果顯示rLZ-8 誘導上皮細胞標誌蛋白和抑制間質細胞標誌蛋白表現，並且顯著性的抑制上皮生長因子誘導之細胞移動現象。另外rLZ-8 抑制上皮間質細胞轉換過程，伴隨著抑制細胞移動的關鍵蛋白-局部粘著斑激酶(FAK)與Src活性；同時也抑制調節Snail表現的重要訊號傳遞路徑:AKT和ERK，進而造成Snail表現量下降。進一步老鼠活體試驗分析，rLZ-8可以同時抑制老鼠肺癌細胞(LLC-1)的轉移與增長。值得注意地，不論在體外或體內試驗，rLZ-8確實顯著且明確地降低表皮生長因子授器蛋白表現量。總括上述，rLZ-8於調節上皮-間質細胞轉化過程重要關鍵為抑制FAK活性以及降低上皮生長因子授器表現。此研究結果提供rLZ-8為未來具有潛能開發為抗人類非小細胞肺癌增長與其轉移的重要新分子藥物。

Ling Zhi-8 (LZ-8) is a human immune-modulatory protein from Ganoderma lucidum. Our recent study demonstrated that LZ-8 effectively can prevent lung cancer cell proliferation; however, the mechanism of recombinant LZ-8 (rLZ-8) exhibits anti-metastasis property is unclear. Epithelial-mesenchymal transition (EMT) process has been regarded as the critical event in tumor metastasis and induced by multi-signaling pathway. The epidermal growth factor receptor (EGFR)-mediated signal transductions play an important role in tumor progression and EMT process. Regulation of Snail, through EGF-induced downstream signaling activation, plays a pivotal role of EMT determinate. We hypothesized that rLZ-8 may suppress the EGFR-driven EMT in human non small cell lung cancer cells (NSCLC). Treatment of lung cancer cells with rLZ-8 induced E-cadherin and inhibited N-cadherin as well as it significantly inhibits EGFR-induced cell motility in vitro. The mechanism of rLZ-8 mediated inhibition of EMT was corroborated by suppressing autophosphorylation of FAK and reduces Src activity. Concomitantly, rLZ-8 also suppresses ERK and AKT downstream pathways and Snail expression. It was further demonstrated that rLZ-8 suppresses tumor growth and metastasis in Lewis lung carcinoma cells-bearing mice. Interesting, rLZ-8 significantly and specifically downregulates EGFR protein level in vitro and in vivo. In conclusion, the role of rLZ-8 in the regulation of EMT was inhibited by downregulation of EGFR level and by suppression of FAK activity. These findings suggest that rLZ-8 may be a potentially anti-metastasis agent in the treatment of human NSCLC.

Abstract..................................................3
中文摘要...................................................4
Introduction..............................................5
Materials and Methods.....................................9
Cell lines................................................9
Preparation of the recombinant Ling Zhi-8 (rLZ-8) protein.9
Reagents and antibodies...................................9
Western blot analysis.....................................10
Nuclear protein extraction................................10
Wound closure assays......................................10
In vitro cell migration and invasion transwell assays.....11
Determination of cell morphology..........................11
In vivo tumor metastasis and growth assay.................11
Statistical analysis......................................12
Results...................................................13
rLZ-8 inhibits proliferation of non-small-cell lung cancer cells.....................................................13
rLZ-8 inhibits the invasive and migratory properties of A549 cell.................................................14
rLZ-8 inhibits migration and invasion of A549 cells in vitro.....................................................14
rLZ-8 downregulates the ERK and AKT signaling pathway.....15
rLZ-8 suppresses autophosphorylation of FAK and reduces Src activity..................................................16
rLZ-8 downregulates Snail and Slug expression via ERK and AKT inhibition............................................16
rLZ-8 inhibits activity of EGFR tyrosine kinase and downregulaes expression of EGFR...........................17
rLZ-8 dramatically reduces the metastatic potential of LLC-1 cells-bearing mice......................................18
Discussion................................................20
References................................................25
Figure Legends............................................34
Figure 1. rLZ-8 inhibits proliferation of A549............34
Figure 2. rLZ-8 suppresses the expression of mesenchymal-like gene products and induces the expression of epithelial-like gene product.........................................35
Figure 3. rLZ-8 suppresses the migratory and invasive properties of A549 cells..................................37
Figure 4. rLZ-8 inhibits EGFR-mediated downstream signaling pathways..................................................40
Figure 5. rLZ-8 inhibits FAK/Src axis signaling pathway...42
Figure 6. rLZ-8 inhibits Snail and Slug expression........44
Figure 7. rLZ-8 modulates EGFR expression by inducing protein degradation.......................................46
Figure 8. rLZ-8 inhibits EMT program via modulates EGFR expression in LLC-1 cells.................................48
Figure 9. rLZ-8 inhibits tumor metastasis in LLC-1-bearing mice......................................................51
Figure 10. rLZ-8 inhibits tumor growth in LLC-1-bearing mice......................................................52
Figure 11. Schematic diagram of the pathways by rLZ-8 contributes in the regulation and interference of the EMT program...................................................54
Supplementary Figure 1. Gefitinib inhibits EGFR-dependent proliferation of A549.....................................55
Supplementary Figure 2. EGFR tyrosine kinase inhibitor, Gefitinib suppresses the migratory property of A549 cells.56
Supplementary Figure 3. rLZ-8 does not affect expression of TGFβR and integrin β1.....................................57
Supplementary Figure 4. rLZ-8 downregulates EGFR in highly migrated cells, CL1-5 and H460............................58
Supplementary Figure 5. rLZ-8 downregulates EGFR in breast cancer cell lines.........................................59

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