以霍氏格里蒙菌熱穩定溶血素結合辨認胜肽之抗癌藥物之應用

Abstract

霍氏格里蒙菌之熱穩定溶血素（Thermostable direct hemolysin, Gh-TDH）已知對於血球和細胞有程度不一的傷害性。目前已證實熱穩定溶血素為四聚體的形式，其N-端胜肽參與熱穩定溶血素和目標膜蛋白之結合，從而引發結合過程後之溶血和細胞毒性。熱穩定溶血素具穿孔毒素之特性，可破壞細胞膜結構並形成孔洞，改變細胞膜內的滲透壓平衡導致細胞死亡。因此我們將溶血素的穿孔毒性應用到治療腫瘤細胞，並改造成能專一性辨認腫瘤細胞之標靶藥物。本實驗室已將單點突變過後，毒性減弱之溶血素單體（Gh-TDHR46E）結合上表皮生長因子受體辨識胜肽（EB）對細胞株做測試，實驗結果顯示此融合蛋白藥物可辨認表現上表皮生長因子受體（EGFR）之A431細胞株，並抑制癌細胞生長。 本論文之研究是將野生型熱穩定溶血素在N-端接上辨認胜肽，使溶血素之溶血和細胞毒性降低，在辨認胜肽上有一蛋白酶切位可被癌細胞分泌之蛋白酶所切除，毒性降低之溶血素被切除N-端辨認胜肽後，將會回復其細胞毒性，並殺死目標癌細胞。首先是結合金屬基質蛋白酶（MMP-7）辨認位之熱穩定溶血素（MMP7-TDHWT），金屬基質蛋白酶之辨認胜肽含有八個胺基酸，其蛋白酶切位在辨認胜肽中央，切除後會殘餘四個胺基酸與熱穩定溶血素鍵結。結果顯示MMP7-TDHWT和野生型熱穩定溶血素（Gh-TDHWT）之溶血活性沒有顯著差異，再以不同金屬基質蛋白酶表現量之細胞株做測試，高表現金屬基質蛋白酶之HT-29細胞株和低表現金屬基質蛋白酶之MDA-MB-231細胞株在細胞毒性測試上，和Gh-TDHWT比較都顯示毒性未如預期般被抑制。其次是結合Furin蛋白辨認位和前列腺專一性抗原（PSA）辨認位之熱穩定溶血素（Furin-PSA-TDHWT），此融合蛋白之辨認胜肽Furin和前列腺專一性抗原辨認位各含有六個胺基酸，可分別辨認Furin和前列腺專一性抗原，並被前列腺專一性抗原所切除，釋放出切除辨認胜肽後之Gh-TDHWT。實驗中又以N端結合六個His胺基酸之Furin-PSA-TDHWT（His-Furin-PSA-TDHWT）做毒性降低測試。結果顯示His-Furin-PSA-TDHWT與Gh-TDHWT比較後之溶血活性成功被降低，而Furin-PSA-TDHWT和His-Furin-PSA-TDHWT對前列腺癌細胞株LNCaP細胞之毒性測試，與Gh-TDHWT相較下也明顯降低。接下來前列腺專一性抗原之酵素活性測試中，將LNCaP細胞株不含血清之培養基液收集並濃縮，以取得LNCaP細胞株分泌之前列腺專一性抗原，和剛純化完之His-Furin-PSA-TDHWT蛋白作用，結果顯示反應三天後蛋白有明顯被切除的現象。這些熱穩定溶血素融合蛋白提供穿孔毒素在癌症治療藥物上更多的發展以及應用。

Thermostable Direct Hemolysin from Grimontia hollisae (Gh-TDH) has been reported as a pore-forming toxin that results in diverse damage to different kinds of cells. Recently, studies have suggested that TDH is the tetrameric protein by nature, which means it can form pores on cell membranes leading to changing the osmotic pressure, disturbing cell homoeostasis and causing cell death. The N-terminal helix of Gh-TDH has indicated the correlation with binging activity on targeted cell membranes, contributing to hemolytic and cytotoxic activity afterwards. Therefore, the pore-forming toxic activity of Gh-TDH has been adjusted for the requisition of cancer treatment as a protein drug. In order to reduce and avoid ubiquitous toxicity of Gh-TDH, we choose to design several targeted fusion proteins under the Gh-TDH backbone for applying this pore-forming toxin in the treatment of few cancers. In previous research, our group indicated when Gh-TDH conjugated with a peptide at the N-terminal region, resulting in the decrease of hemolytic and cytotoxic activity. Furthermore, construction of mutant Gh-TDHR46E (monomeric form) with a targeting peptide such as epidermal growth factor receptor (EGFR) binding peptide (EB) to obtain a fusion protein, having significant anti-tumor effects on A431 cells1. In my study, there are several Gh-TDH recombinant proteins are designed as the pro-drug by using the concepts of recognition peptide to cell membrane and/or the cleavage sequences for cellular protease activation. The selective target molecules for the designs are MMP-7, Furin and PSA. The recognition peptide is conjugated at the N-terminus of Gh-TDH which might cause toxicity decrease of the designed recombinant protein. Besides, there is a cleavage site at the recognition peptide, which can cleave by proteolytic proteins produced by cancer cells. Next, the recognition peptide contacts with the target cancer cells with the treatment of recombinant protein. After that, the proteolytic proteins secreted from cancer cells interact with recognition peptide resulting in excision at cleavage site. Gh-TDH resumes wild-type activity after peptide-conjugated TDH releases sections cleaved by enzymatic proteins. Afterwards, active Gh-TDH can cause nearby cancer cell death as expected. The protein MMP7-TDHWT is the wild-type Gh-TDH (Gh-TDHWT) conjugates with eight amino acids of MMP-7 recognition and cleavage peptide. MMP7-TDHWT in comparison with Gh-TDHWT shows no significant difference of hemolytic and cytotoxic activity. Furin-PSA-TDHWT which is the Gh-TDHWT conjugates with six amino acids of Furin recognition peptide, and six amino acids of PSA cleavage site peptide. His-Furin-PSA-TDHWT is Furin-PSA-TDHWT conjugates with six His amino acids at the N-terminus. The result of hemolytic activity of His-Furin-PSA-TDHWT is weaker than Gh-TDHWT significantly. And the MTT assay suggests that cytotoxicity of Furin-PSA-TDHWT for LNCaP prostate cancer cells is weaker than Gh-TDHWT. However, the cytotoxicity of His-Furin-PSA-TDHWT is much weaker than Furin-PSA-TDHWT. The conditioned medium of LNCaP cells incubates with His-Furin-PSA-TDHWT for more than three days, which shows the enzymatic activity of PSA. The result indicates releasing of Gh-TDHWT from His-Furin-PSA-TDHWT by enzymatic cleavage.