近年來，可毒殺癌細胞而不傷害正常細胞的抗菌胜肽，其抗癌的潛力越來越受到重視與投入。本實驗室從Lactobacillus casei ATCC 334菌株的基因序列篩選出具有抗菌活性的胜肽m2163。在過去的研究發現，m2163可以抑制人類大腸癌細胞SW480生長，但其中的機制及路徑尚不清楚。本實驗關注於m2163對SW480細胞造成的細胞凋亡及細胞壞死現象，除了透過抑制細胞凋亡的caspases inhibitor Z-VAD-FMK和抑制細胞壞死的RIPK1 inhibitorNecrostain-1分析m2163對這兩種路徑的使用情形，也試著由此結果探討更多m2163殺害細胞的可能機制。實驗內容包括以MTT assay測試m2163對SW480的抑制效果，Annexin V-FITC/PI雙染實驗觀察細胞凋亡和壞死的現象，核酸染劑PI測試細胞膜通透性的改變，自由基指示劑DCFDA檢測活性氧類 (ROS) 的生成，螢光標記追蹤m2163在細胞中的位置。實驗結果確認m2163會促使SW480發生細胞凋亡及細胞壞死，且可能隨m2163使用劑量有比例上的變化，並推測m2163的作用機制裡有caspases和RIPK1相關以外的路徑存在。ROS的生成雖然可能幫助殺害細胞，但與細胞凋亡和壞死關係不明顯。m2163多與SW480細胞膜結合，胞內則尚無法辨別。

In recent years, the anticancer potential of antimicrobial peptides (AMPs), which exhibit toxicity toward eukaryotic cancer cells but not to normal mammalian cells, has received attention and investment. Previously, our laboratory screened a novel anticancer AMP named m2163 from Lactobacillus casei ATCC 334. M2163 was confirmed to suppress human colorectal cancer cell line SW480 growth, but the mechanism and pathway are not well understood. This study focuses on the apoptotic and necrotic effects of m2163 in SW480 cell. The caspases inhibitor Z-VAD-FMK as apoptosis inhibitor and RIPK1 inhibitor Necrostain-1 as necrosis inhibitor were used to analyze how m2163 utilize the two processes, and then also try to find out more possible anticancer mechanism of m2163. We used MTT assay to test the proliferation inhibition of m2163 in SW480 cell, Annexin V-FITC/PI double staining for observing induced apoptosis and necrosis, nucleic acid dye PI for testing the change of cell membrane permeability, free-radical indicator DCFDA for detecting the generation of ROS, fluorescein labeling for cellular localization. The experiment results revealed that m2163 can induce apoptosis and necrosis in SW480 cell and the proportion may vary with dosage. Furthermore, there are pathways not associated with caspases and RIPK1 in the anticancer mechanism of m2163. The generation of ROS may help killing cells, but the connection with apoptosis and necrosis is not obvious. M2163 generally attach on SW480 cell membrane. As for cell cytoplasm, it is not clear yet.

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