Integrative bioinformatics and proteomics-based discovery of an eEF2K inhibitor (cefatrizine) with ER stress modulation in breast cancer cells.

Eukaryotic elongation factor-2 kinase (eEF2K), a unique calcium/calmodulin-dependent protein kinase, is well known to regulate apoptosis, autophagy and ER stress in many types of human cancers. Therefore, eEF2K would be regarded as a promising therapeutic target; however, the eEF2K-regulated mechanism and its targeted inhibitor still remain to be discovered in cancer. Herein, we constructed a protein-protein interaction (PPI) network of eEF2K and achieved an eEF2K-regulated ER stress subnetwork by bioinformatics prediction. Then, we found that the differential protein expressions involved in ER stress in the context of si-eEF2K-treated MCF-7 and MDA-MB-436 cells by iTRAQ-based analyses, respectively. Integrated into these aforementioned results, we constructed a core eEF2K-regulated ER stress subnetwork in breast cancer cells. Subsequently, we screened a series of candidate compounds targeting eEF2K and discovered a novel eEF2K inhibitor (cefatrizine) with an anti-proliferative activity toward breast cancer cells. Moreover, we found that cefatrizine induced ER stress in both MCF-7 and MDA-MB-436 cells. Interestingly, we demonstrated that the mechanism of cefatrizine-induced ER stress was in good agreement with our bioinformatics and proteomics-based results. In conclusion, these results demonstrate that a novel eEF2K inhibitor (cefatrizine) induces ER stress in breast cancer cells by integrating bioinformatics prediction, proteomics analyses and experimental validation, which would provide a clue for exploring more mechanisms of eEF2K and its targeted inhibitors in cancer therapy.

Sfrp5 expression and secretion in adipocytes are up-regulated during differentiation and are negatively correlated with insulin resistance.

We have examined the patterns of Sfrp5 (secreted frizzled-related protein 5) mRNA expression and protein secretion during adipocyte differentiation, and investigated the potential role of Sfrp5 in IR (insulin resistance) in adipocytes. 3T3-L1 pre-adipocytes were induced for differentiation, and RT-PCR (reverse transcription-PCR) and ELISA assays were used to determine Sfrp5 mRNA expression and protein secretion. The results showed that with the differentiation and maturity of pre-adipocytes, transcription and protein secretion of Sfrp5 gradually increased, peaking on the 9th day of differentiation. Sfrp5 mRNA expression in mature adipocytes was decreased by 20, 22 and 32 upon treatment with dexamethasone, insulin and TNF (tumour necrosis factor) respectively, whereas Sfrp5 protein secretion was decreased by 15, 17 and 30%, correspondingly. In contrast, Sfrp5 mRNA expression in mature adipose was increased by 34 and 19% upon treatment with rosiglitazone and metformin respectively, whereas Sfrp5 protein secretion was increased by 10 and 6%, correspondingly. In conclusion, Sfrp5 mRNA expression and protein secretion depend on the differentiation of adipocytes. The dysregulation of Sfrp5 expression and secretion is directly correlated with IR. Up-regulation of Sfrp5 expression and secretion in adipocytes may be one crucial mechanism by which rosiglitazone and metformin improve insulin sensitivity.