Collapsin response mediator protein-1與穿胞胜肽接合體之製備及其於肺腺癌細胞之胞內運輸研究

Abstract

Collapsin response mediator protein-1 (CRMP-1) 是一種能促進神經細胞生長椎崩塌 (growth cone collapse) 及軸突誘導 (axonal guidance) 的蛋白質，CRMP-1和癌細胞的侵襲能力也具有相關性。CL1-5是具有高度侵襲能力的人類肺腺癌細胞，其細胞內CRMP-1的表現量較侵襲能力低的CL1-0少，以基因轉染 (gene transfection) 的方式增加CL1-5的CRMP-1表現量，可抑制其侵襲能力並減少絲狀偽足的產生。肝素結合凝血附著素 (heparin-binding haemagglutinin adhesin，HBHA) 是從結核桿菌 (Mycobacterium tuberoculosis) 中發現的蛋白質，其C端區域是與宿主肺部表皮細胞黏合的重要區段，此段結構主要是由兩個片段重覆排列而成：KKAAPA (R1) 和KKAAAKK (R2)。過去我們發現以1R1 + 2R2 方式組合而成的胜肽HBHAc，能成功地攜帶重組精胺酸去亞胺酶 (recombinant arginine deiminase，rADI) 進入人類乳癌細胞 (MCF-7)。為了增加CL1-5內CRMP-1的量，本研究利用大腸桿菌 (Escherichia coli) 所製造出重組之CRMP-1 (rCRMP-1)，以高通量篩選的方式尋找rCRMP-1的再摺疊條件，接著透過化學修飾接上HBHAc，預期rCRMP-1能夠經由HBHAc的幫助進入到CL1-5。 首先，我們利用Ni-NTA管柱以梯度沖提的方式將rCRMP-1從包涵體中純化出來，接著以高通量篩選的方式找出rCRMP-1摺疊的最佳條件，依此條件進行rCRMP-1的透析與再摺疊，透析完成後，將rCRMP-1標記上FITC及接上HBHAc。細胞實驗部分，我們選擇CL1-5作為模式細胞株，以SRB assay及MTT assay測試HBHAc-rCRMP-1是否會影響CL1-5的存活率，接著，以流式細胞儀分析HBHAc-CRMP-1進入細胞的效率，確認HBHAc-rCRMP-1能有效地進入細胞後，再以F-actin 染色試驗來評估HBHA-rCRMP-1的活性。 研究結果顯示，本研究的純化方法能成功地純化出高純度的rCRMP-1，我們找出50 mM Tris、0.1% 甘露醇 (pH 9.0) 為rCRMP-1再摺疊的最佳條件，透析完成後我們將rCRMP-1置換到1X PBS (pH 9.0) 的環境下，並順利地將rCRMP-1標記FITC及接上HBHAc，我們發現以「先接HBHAc，再接FITC」的順序反應，可使得rCRMP-1及HBHAc-rCRMP-1的螢光量較為一致。在細胞存活率部分，SRB assay結果顯示HBHAc-rCRMP-1及rCRMP-1皆不會造成細胞毒性，但投予HBHAc-rCRMP-1會使得CL1-5的型態產生改變；而利用MTT assay會得到偽陽性的結果，無法準確評估細胞毒性。在細胞活性試驗的部分，我們投予不同劑量的HBHAc-rCRMP-1及rCRMP-1，對F-actin進行螢光染色觀察，結果顯示CL1-5細胞投予30 nM和100 nM的HBHAc-rCRMP-1後，其細胞帶有絲狀偽足的比率有減少的趨勢。在細胞攝取實驗，以EGFP作為報導可觀察到HBHAc能有效地將EGFP送入CL1-5，證明HBHAc在CL1-5具有遞送的功能；接著我們投予300 nM的rCRMP-1及HBHAc-rCRMP-1，培養兩小時後，HBHAc-rCRMP-1與rCRMP-1進入細胞的比率皆高於對照組 (p<0.05)，但兩者間的差異在統計上並不顯著；我們同樣投予300 nM的HBHAc-rCRMP-1與rCRMP-1至MCF-7，HBHAc-rCRMP-1進入細胞的比率約為30%，rCRMP-1的比率為6%，然而我們需要更多的結果去確認HBHAc-rCRMP-1是否在CL1-5與MCF-7的細胞攝取有所差異。 本研究成功地純化出rCRMP-1、找到rCRMP-1摺疊的適當條件，並且製備出HBHAc-rCRMP-1的接合體，但PBS的環境並不適於rCRMP-1的保存。HBHAc-rCRMP-1會使CL1-5的型態產生改變並減少CL1-5細胞絲狀偽足的表現量，卻不會影響其細胞存活率。HBHAc能成功地遞送EGFP及rCRMP-1到CL1-5細胞，且HBHAc遞送rCRMP-1的能力會因細胞株不同而有所差異。而我們因為rCRMP-1的來源有限，利用化學鍵結的方式結合穿胞胜肽與蛋白質雖是有效地蛋白質遞送策略，但未來我們需要更多的結果去確認。

Collapsin response mediator protein-1 (CRMP-1) is a protein inducing growth cone collapse and axonal guidance of neural cells. It is also related to the invasive ability of cancer cells. CL1-5 cells are human lung adenocarcinoma cells with highly invasive ability compared to CL1-0 cells. The expression of CRMP-1 in CL1-5 is lower than that in CL1-0. The increase of the expression of CRMP-1 in CL1-5 by gene transfection inhibited invasive activity and reduced the formation of filopodia. Heparin-binding haemagglutinin adhesion (HBHA) is a protein discovered from Mycobacterium tuberoculosis. Its C-terminal domain is an important part to adhere pulmonary epithelial cells of host, and this domain is composed of two repeated motifs : KKAAPA motif (R1) and KKAAAKK (R2). In our previous study, the peptide with the sequence of 1R1 +2R2 named HBHAc successfully carried recombinant arginine deiminase (rADI) into human breast cancer cells (MCF-7) intracellulatly. In order to increase the intracellular amount of CRMP-1 in CL1-5, this study used the recombinant CRMP-1 (rCRMP-1) produced from Escherichia coli and found the best refolding condition of rCRMP-1 with a high-throughput screening method. The next, the rCRMP-1 was conjugated with HBHAc by chemical modification, and further confirmed the biological activity of HBHAc modified rCRMP-1 in CL1-5. First, we used the nickel-nitrilotriacetic acid (Ni-NTA) column to purify rCRMP-1 from inclusion bodies with gradient elution. We found the best refolding condition with a high-throughput screening method and chose this condition to dialyze and refold the rCRMP-1. After the completion of dialysis, the rCRMP-1 was labeled with FITC and linked with HBHAc. In cell experiments, we choose CL1-5 as a model cell line, and used SRB assay and MTT assay to investigate whether HBHAc-rCRMP-1 affect the cell viability. We subsequently used flow cytometry to analyze the uptake efficiency of HBHAc-rCRMP-1, after confirming HBHAc-rCRMP-1 could effectively enter the cells, we evaluated the activity of HBHAc-rCRMP-1 by actin staining assay. We obtained the rCRMP-1 from the inclusion bodies with a highly purity, we also found the best buffer (50 mM Tris, 0.1% mannitol, pH 9.0) condition for rCRMP-1 refolding. After dialysis, we changed the buffer into 1X PBS (pH 9.0) and rCRMP-1 was labeled with FITC and linked with HBHAc. In cell viability experiment, the SRB assay results showed both HBHAc-rCRMP-1 and rCRMP-1 didn’t cause the cytotoxicity, but the cell morphology of CL1-5 was changed after treating with HBHAc-rCRMP-1. In contrast, MTT assay showed a false-positive result because of some interference released from cells. In actin staining assay, we treated the cells with different dose of proteins, then we stained the F-actin to observe its distribution. The results showed the percentage of CL1-5 cells with filopodia was reduced after treating with 30 nM and 100 nM of HBHAc-rCRMP-1. In cellular uptake study, we used enhanced green fluorescence protein (EGFP) as a reporter to observe the delivery ability of HBHAc, and HBHAc could carry EGFP into CL1-5. We further treated CL1-5 with 300 nM of HBHAc-rCRMP-1 and rCRMP-1, after 2 hours incubation, the percentage of fluorescence positive cells of both treatment group was higher than that of control group (p<0.05), but the differences between them were not statistically significant. We also treated MCF-7 with the same dose of proteins, the HBHAc-rCRMP-1 group showed 30% of fluorescence positive cells, but only 6% in rCRMP-1 group. However, we need further results to confirm that whether HBHAc-rCRMP-1 had different uptake efficiency in different cell lines. In conclusion, we successfully purified the rCRMP-1, found the suitable condition for rCRMP-1 refolding, and prepared the HBHAc-rCRMP-1 conjugates. The PBS was not an appropriate buffer for rCRMP-1 perservation. HBHAc-rCRMP-1 changed the cell morphology and reduced the expression of filopodia in CL1-5 cells, but not affected the cell viability. HBHAc had the ability to carry EGFP and rCRMP-1 into CL1-5 cells, and HBHAc-rCRMP-1 showed different uptake efficiency in CL1-5 and MCF-7 cells. However due to the limited availability of rCRMP-1, the conjugation of cell-penetrating peptides and proteins may be a useful protein delivery strategy, we need to confirm the results in the future.