Epitelial-to-mesenchimal transition and invasion are upmodulated by tumor-expressed granzyme B and inhibited by docosahexaenoic acid in human colorectal cancer cells.
Epitelial-to-mesenchimal transition and invasion are upmodulated by tumor-expressed granzyme B and inhibited by docosahexaenoic acid in human colorectal cancer cells.
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DOI：
10.1186/s13046-016-0302-6
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发表时间：
2016-02-02
期刊：
影响因子：
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通讯作者：
Velotti F
中科院分区：
文献类型：
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作者：
D'Eliseo D;Di Rocco G;Loria R;Soddu S;Santoni A;Velotti F
颗粒酶 B (GrB) 是一种丝氨酸蛋白酶，传统上被认为由细胞毒性淋巴细胞表达，可诱导靶细胞凋亡。然而，GrB 也由多种正常细胞和肿瘤细胞产生，并可能作用于多个靶标，因此逐渐出现，它可能代表了广泛的基本生物过程的强大调节剂。我们之前已经表明，GrB 在尿路上皮癌组织中表达，其表达与病理性肿瘤扩散和 EMT 相关。我们还表明，二十二碳六烯酸（DHA）是一种具有抗肿瘤活性的膳食 ω-3 多不饱和脂肪酸，在抑制尿路上皮和胰腺癌细胞侵袭的同时，还可以在体外抑制其 GrB 表达。在这项研究中，我们对一组具有不同侵袭能力的结直肠癌 (CRC) 细胞进行了 GrB 表达以及 GrB 对其 EMT 和侵袭表型的贡献进行了表征。此外，我们还研究了 DHA 对 CRC 细胞相关 GrB 表达、EMT 和侵袭的影响。通过Western blotting评估GrB和EMT相关标志物的表达水平。 GrB 敲低是通过 Stealth RNAi 小干扰 RNA 沉默进行的，并通过转染人 GrB 载体进行异位 GrB 表达。通过BioCoat Matrigel 侵袭室测试测定细胞侵袭。 GrB 在 57.1% CRC 细胞系和 100% CRC 衍生的癌症干细胞中产生。尽管 GrB 在侵袭性和非侵袭性 CRC 细胞中均呈组成型表达，但侵袭性 CRC 细胞中 GrB 的缺失会下调其体外侵袭性，表明 GrB 对 CRC 侵袭性有贡献。根据对三种 EMT 生物标志物（Snail1、E-钙粘蛋白、N-钙粘蛋白）的癌细胞表达的分析，GrB 功能的丧失或获得分别下调或上调 EMT。此外，TGF-β1驱动的EMT与CRC细胞系中GrB表达的增强有关，而GrB的缺失导致TGF-β1驱动的EMT下调。此外，DHA 在体外抑制 CRC 细胞的 GrB 表达、EMT 和侵袭。这些发现展示了 GrB 作为 CRC 细胞 EMT 上调剂的新作用。此外，这些结果支持使用 DHA（一种无毒性作用的膳食化合物）作为 CRC 治疗的辅助剂。
Granzyme B (GrB) is a serine protease, traditionally known as expressed by cytotoxic lymphocytes to induce target cell apoptosis. However, it is emerging that GrB, being also produced by a variety of normal and neoplastic cells and potentially acting on multiple targets, might represent a powerful regulator of a wide range of fundamental biological processes. We have previously shown that GrB is expressed in urothelial carcinoma tissues and its expression is associated to both pathological tumor spreading and EMT. We have also shown that docosahexaenoic acid (DHA), a dietary ω-3 polyunsaturated fatty acid with anti-tumor activity, while inhibiting urothelial and pancreatic carcinoma cell invasion also inhibited their GrB expression in vitro. In this study, we characterized a panel of colorectal carcinoma (CRC) cells, with different invasive capabilities, for GrB expression and for the contribution of GrB to their EMT and invasive phenotype. In addition, we investigated the effect of DHA on CRC cell-associated GrB expression, EMT and invasion. The expression levels of GrB and EMT-related markers were evaluated by Western blotting. GrB knockdown was performed by Stealth RNAi small interfering RNA silencing and ectopic GrB expression by transfection of human GrB vector. Cell invasion was determined by the BioCoat Matrigel invasion chamber test. GrB was produced in 57.1 % CRC cell lines and 100 % CRC-derived Cancer Stem Cells. Although GrB was constitutive expressed in both invasive and noninvasive CRC cells, GrB depletion in invasive CRC cells downmodulated their invasion in vitro, suggesting a contribution of GrB to CRC invasiveness. GrB loss or gain of function downmodulated or upmodulated EMT, respectively, according to the analysis of cancer cell expression of three EMT biomarkers (Snail1, E-cadherin, N-cadherin). Moreover, TGF-β1-driven EMT was associated to the enhancement of GrB expression in CRC cell lines, and GrB depletion led to downmodulation of TGF-β1-driven EMT. In addition, DHA inhibited GrB expression, EMT and invasion in CRC cells in vitro. These findings present a novel role for GrB as upmodulator of EMT in CRC cells. Moreover, these results support the use of DHA, a dietary compound without toxic effects, as adjuvant in CRC therapy.