晚期结直肠癌（CRC）的治疗取决于正确选择个性化策略。HAMLET（致癌细胞致死的人类α-乳白蛋白）是一种天然的蛋白脂复合物，可能成为一种新型的癌症预防和治疗候选物。我们的目的是研究HAMLET对不同KRAS /BRAF突变状态的CRC细胞的存活率、死亡途径和线粒体生物能的影响。我们使用HAMLET处理了三个细胞系（Caco-2、LoVo、WiDr），评估细胞代谢活性和存活率、流式细胞术中的凋亡和坏死细胞、促凋亡和抗凋亡基因以及蛋白质表达。线粒体呼吸（氧消耗）速率通过高分辨率呼吸测定系统Oxygraph-2k进行记录。HAMLET复合物对所有调查的CRC细胞系都具有细胞毒性，且这种作用是不可逆的。流式细胞术表明，HAMLET诱导坏死性细胞死亡，同时略微增加了凋亡细胞的比例。 WiDr细胞的代谢、克隆形成、坏死/凋亡水平和线粒体呼吸明显受到的影响要小于其他细胞。HAMLET以剂量依赖方式对人类CRC细胞表现出不可逆的细胞毒性，导致坏死性细胞死亡并抑制外部凋亡途径。BRAF突变的细胞系比其他类型的细胞系更具有抗药性。HAMLET减少了Caco-2和LoVo细胞系的线粒体呼吸和ATP合成，但不影响WiDr细胞的呼吸。癌细胞经HAMLET预处理对线粒体外膜和内膜通透性没有影响。 ©2023年。作者（们）。

Treatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.We treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.The HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.HAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells' respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.© 2023. The Author(s).