中文摘要

蛇毒中的L-氨基酸氧化酶（svLAAOs）是一類含有醣基的黃素酶，被認為在癌症治療上深具潛力，其主要的細胞毒性源自於這些酵素與L-氨基酸基質進行反應時所產生的過氧化氫（H2O2）。然而，在svLAAO治療下，癌細胞除了被殺死之外，它是如何適應H2O2引起的氧化壓力，尚未被研究。在本研究中，我們純化且確認了來自Naja kaouthia毒液的svLAAO（NK-LAAO）的性質，同時研究人類肺腺癌A549細胞在NK-LAAO治療下的整體反應。親緣演化分析顯示svLAAOs分為兩個主要的演化支，即眼鏡蛇科和蝮蛇科的svLAAOs，其中NK-LAAO來自眼鏡蛇科svLAAOs，且與眼鏡蛇科Naja atra（NA-LAAO）的svLAAO最相關。此外，對svLAAOs活性位點相關殘基的分析結果顯示，在蝮蛇科svLAAOs中高度保守的His223，在眼鏡蛇科svLAAOs中則不高度保守，這是之前被提出具有酶活性的關鍵催化殘基。在結構上，NK-LAAO模型顯示為功能性二聚體，每個protomer由三個區域（基質結合區域，黃素腺嘌呤二核苷酸結合區域和螺旋區域）和三個潛在的N-連接醣化位點（N172、N194和N359）組成。NK-LAAO對疏水性L-氨基酸，如L-Trp、L-Met和L-Leu有顯著的基質偏好，對鹼性L-氨基酸L-Arg有適中的偏好，對酸性L-氨基酸L-Glu的特異性明顯較低。重要的是，NK-LAAO具有顯著的細胞毒性活性，依賴於培養基中的L-氨基酸組成，而不依賴於酵素表面的N-連接醣化，通過誘發A549癌細胞中的細胞外過氧化氫（H2O2）和細胞內活性氧（ROS）引起的細胞死亡。令人出乎意料，我們發現一種癌細胞部署的耐受機制，用以減緩NK-LAAO的抗癌活性。該酶的治療通過增加Pannexin 1（Panx1）驅動的細胞內鈣離子（iCa2+）升高，並活化PI3K/Akt信號傳導途徑，放大癌細胞的白細胞介素（IL）-6表達，使其能夠逃避氧化壓力誘導的細胞死亡，獲得上皮間葉轉化（EMT）樣的表現，並具有轉移能力。因此，shRNA介導的IL-6表達耗盡使癌細胞對NK-LAAO誘導的氧化壓力敏感，並抑制了NK-LAAO驅動的細胞遷移和侵襲。總而言之，我們的研究強調在使用svLAAOs進行癌症治療時需要謹慎，並建議以增強Panx1/iCa2+/IL-6軸作為提高svLAAOs基礎抗癌療法有效性的方法。

ABSTRACT

Snake venom L-amino acid oxidases (svLAAOs) are glycosylated flavoenzymes that have been proposed as promising candidates for anticancer therapeutics based on the primary cytotoxicity of hydrogen peroxide (H2O2) generated during reactions of the enzymes with their L-amino acid substrates. However, how cancer cells adapt to H2O2-induced oxidative stress other than to be killed under the context of svLAAO treatment has not been investigated. In this study, we purify and characterize the properties of a svLAAO from the Elapid snake Naja kaouthia venom (NK-LAAO), and comprehensively investigate the overall responses of human lung adenocarcinoma A549 cells under the treatment of NK-LAAO. Phylogenetic analysis reveals that svLAAOs split into two major clades of elapid and viperid svLAAOs, of which NK-LAAO arises from elapid svLAAOs and is the most closely related to the svLAAO from the Elapid snake Naja atra (NA-LAAO). In addition, analysis of the active site-related residues of svLAAOs uncovered that His223, a previously proposed critical catalytic residue for the enzyme activity, is highly conserved in the viperid but not the elapid svLAAOs. Structurally, the NK-LAAO model shows a functional dimer with each protomer consisting of three domains (substrate-binding, FAD-binding, and helical domains) and three putative N-linked glycosylation sites (N172, N194, and N359). NK-LAAO has a significantly high substrate preference for the hydrophobic L-amino acids L-Trp, L-Met, and L-Leu, a moderate preference for the basic L-amino acid L-Arg, and a significantly low specificity for the acidic L-amino acid L-Glu. Importantly, NK-LAAO exhibits substantial cytotoxic activity, which depends on the L-amino acid composition in the culture medium but not the N-linked glycans on the enzyme surface, by triggering extracellular hydrogen peroxide (H2O2)- and intracellular reactive oxygen species (ROS)-mediated cell death in A549 cancer cells. Unexpectedly, we explore a tolerant mechanism deployed by cancer cells to impair the anticancer activities of NK-LAAO. The treatment of the enzyme exaggerates interleukin (IL)-6 expression via increasing Pannexin 1 (Panx1)-driven intracellular calcium (iCa2+) elevation and activating the PI3K/Akt signaling pathway to enable cancer cells to evade oxidative stress-induced cell death, acquire epithelial-mesenchymal transition (EMT)-like phenotypes, and gain metastatic abilities. As a result, short hairpin (sh)RNA-mediated depletion of IL-6 expression sensitizes cancer cells to NK-LAAO-induced oxidative stress and inhibits NK-LAAO-driven cell migration and invasion. Taken together, our study urges caution when using svLAAOs in cancer treatment and suggests targeting the Panx1/iCa2+/IL-6 axis as an approach to enhance to improve the effectiveness of svLAAOs-based anticancer therapies.

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