Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.
Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.
复制标题
通过蛋白质印迹评估癌细胞中炎症细胞因子的细胞质和细胞核水平。
DOI：
10.1007/978-1-4939-0928-5_25
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Vancurova,Ivana
中科院分区：
文献类型：
--
作者：
Gatla,HimavanthR;Singha,Bipradeb;Persaud,Valerie;Vancurova,Ivana
炎性细胞因子、白细胞介素-8（IL-8; CXCL 8）和高迁移率族蛋白-1（HMGB 1）的表达和细胞释放增加与癌症进展期间细胞增殖、血管生成和转移增加相关。在前列腺癌和卵巢癌细胞中，IL-8和HMGB 1水平升高与预后不良相关。我们最近发现硼替佐米（BZ）抑制蛋白酶体可以特异性地增加转移性前列腺癌和卵巢癌细胞释放IL-8。在这一章中，我们描述了一个协议，分析细胞质和细胞核水平的IL-8和HMGB 1在前列腺癌和卵巢癌细胞免疫印迹。IL-8定位于两种细胞类型的细胞质中，并且其蛋白水平被BZ显著增加。相比之下，HMGB 1位于细胞核中，BZ仅在卵巢癌细胞中增加其核水平。该方案包括分离细胞质和细胞核提取物，然后进行SDS电泳和蛋白质印迹，并且可以容易地修改以分析其他细胞类型中的细胞质和细胞核细胞因子水平。
Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.