Background/Aims: Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism. Methods: Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting. Results: We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO. Conclusion: Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO.

中文翻译：

---

棕褐色墨水寡肽通过线粒体途径诱导肺癌细胞凋亡

背景/目的：我们先前的研究提出了棕褐色墨水寡肽（SIO）的抗肿瘤活性。在这里，我们试图研究潜在的分子机制。方法：通过细胞计数试剂盒8（CCK-8）分析评估细胞增殖。通过膜联蛋白V /碘化丙啶（PI）染色确定细胞凋亡。线粒体途径的特征在于对Bcl-2，Bax，Caspase-9和Cyto-C的定量分析。通过确定Fas，Caspase-8和NIK分析死亡受体途径。通过测量CHOP，Caspase-12，GRP78和钙蛋白酶的表达来确定内质网（ER）依赖性途径。通过RT-PCR定量相关的基因表达，并通过免疫印迹确定蛋白质水平。结果：我们证明了使用结构改良的SIO（CSIO，5 µM）可显着抑制肺癌细胞A549的细胞增殖并诱导其凋亡。CSIO处理可刺激线粒体途径，死亡受体途径和内质网应激诱导的细胞凋亡。分别施用包括Midiv-1（50 µM，2 h），PDTC（20 µM，PDTC，30 min）和ALLN（20 mM ALLN，5 h）的抑制剂可以轻易逆转CSIO的凋亡诱导作用。结论：我们的数据表明CSIO能够诱导肺癌细胞株的凋亡，这是由所有三种经典的凋亡途径介导的。我们的结果值得对CSIO的抗肿瘤潜力进行进一步的体内研究。分别施用包括Midiv-1（50 µM，2 h），PDTC（20 µM，PDTC，30 min）和ALLN（20 mM ALLN，5 h）的抑制剂可以轻易逆转CSIO的凋亡诱导作用。结论：我们的数据表明CSIO能够诱导肺癌细胞株的凋亡，这是由所有三种经典的凋亡途径介导的。我们的结果值得对CSIO的抗肿瘤潜力进行进一步的体内研究。分别施用包括Midiv-1（50 µM，2 h），PDTC（20 µM，PDTC，30 min）和ALLN（20 mM ALLN，5 h）的抑制剂可以轻易逆转CSIO的凋亡诱导作用。结论：我们的数据表明CSIO能够诱导肺癌细胞株的凋亡，这是由所有三种经典的凋亡途径介导的。我们的结果值得对CSIO的抗肿瘤潜力进行进一步的体内研究。