Valiyari Samira, Salami Mona, Mahdian Reza, Shokrgozar Mohammad Ali, Oloomi Mana, Mohammadi Farsani Azadeh, Bouzari Saeid
Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran.
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran.
Cancer Chemother Pharmacol. 2017 Sep;80(3):451-459. doi: 10.1007/s00280-017-3370-1. Epub 2017 Jun 26.
Interleukin-24 (IL-24) is a unique cytokine in the IL-10 family that reveals tumor-suppressive activity against a broad range of cancers. Alternative splicing of human IL-24 generates several isoforms with different pro-apoptotic activities. In the current study, we aimed to investigate the cytotoxic properties of a recombinant smallest isoform of IL-24 (sIL-24) and the underlying molecular mechanisms in PC-3, A549, U937, and Raji cancer cells as well as normal cell line MRC-5.
Following treatment of the cells with recombinant sIL-24 peptide and full-length IL-24 protein, cytotoxicity was determined by MTT assay. Apoptosis induction was evaluated using annexin-V/PI double staining flow cytometry and Hoechst 33342 staining. The expression of Bax, Bcl-2, cytochrome c, and caspase-3 was analyzed by Western blotting.
MTT assay exhibited that sIL-24 dose and time dependently inhibited the proliferation of IL-24 receptor-positive PC-3, U937, and Raji cells more effectively than full-length IL-24. In contrast, sIL-24 had little cytotoxic effect on A549 cells lacking the IL-24 receptor, or on MRC-5 normal cells. Flow cytometric analysis and morphological observation revealed an efficient apoptosis induction in the receptor-positive cells. Furthermore, Western blot assay demonstrated that cell death induced by sIL-24 was associated with upregulation of the Bax/Bcl-2 ratio, cytochrome c release, and the expression of cleaved caspase-3, suggesting that sIL-24 induced apoptosis mainly through the mitochondrial pathway. Notably, among the tested cells, induction of apoptosis was more significant in PC-3 cells.
Our results suggest that the sIL-24 peptide is a promising candidate for potential treatment of human cancers.
白细胞介素-24（IL-24）是白细胞介素-10家族中的一种独特细胞因子，对多种癌症具有肿瘤抑制活性。人IL-24的可变剪接产生了几种具有不同促凋亡活性的异构体。在本研究中，我们旨在研究重组最小异构体IL-24（sIL-24）在PC-3、A549、U937和Raji癌细胞以及正常细胞系MRC-5中的细胞毒性特性及其潜在分子机制。
用重组sIL-24肽和全长IL-24蛋白处理细胞后，通过MTT法测定细胞毒性。使用膜联蛋白-V/PI双染流式细胞术和Hoechst 33342染色评估凋亡诱导情况。通过蛋白质印迹法分析Bax、Bcl-2、细胞色素c和半胱天冬酶-3的表达。
MTT法显示，sIL-24剂量和时间依赖性地比全长IL-24更有效地抑制IL-24受体阳性的PC-3、U937和Raji细胞的增殖。相比之下，sIL-24对缺乏IL-24受体的A549细胞或MRC-5正常细胞几乎没有细胞毒性作用。流式细胞术分析和形态学观察显示受体阳性细胞中凋亡诱导有效。此外，蛋白质印迹分析表明，sIL-24诱导的细胞死亡与Bax/Bcl-2比值上调、细胞色素c释放以及裂解的半胱天冬酶-3的表达有关，表明sIL-24主要通过线粒体途径诱导凋亡。值得注意的是，在测试的细胞中，PC-3细胞中凋亡诱导更为显著。
我们的结果表明，sIL-24肽是人类癌症潜在治疗的有希望的候选者。
