Quantitative proteomics characterization on the antitumor effects of isodeoxyelephantopin against nasopharyngeal carcinoma.

Isolated from Elephantopus scaber L., a Chinese medicinal herb that is widely used to prevent and treat cancers in China, isodeoxyelephantopin (ESI) exerted antitumor effects on several cancer cells. However, its antitumor mechanism is still not clear. In this study, we found that ESI could induce G2/M arrest and subsequently stimulate cell apoptosis in dose- and time-dependent manners. We used SILAC quantitative proteomics to identify ESI-regulated proteins in cancer cells, and found that 124 proteins were significantly altered in expression. Gene ontology and Ingenuity Pathway Analysis revealed that these proteins were mainly involved in the regulation of oxidative stress and inflammation response. Functional studies demonstrated that ESI induced G2/M arrest and apoptosis by inducing ROS generation, and that antioxidant N-acetyl-l-cysteine could block the ESI-induced antitumor effects. Accumulated ROS resulted in DNA breakage, subsequent G2/M arrest and mitochondrial-mediated apoptosis. ESI upregulated the expression of anticancer inflammation factors IL-12a, IFN-α, and IFN-ß through ROS-dependent and independent pathways. The current work reveals that ESI exerts its antitumor effects through ROS-dependent DNA damage, mitochondrial-mediated apoptosis mechanism and antitumor inflammation factor pathway.

Protective effects of andrographolide analogue AL-1 on ROS-induced RIN-mß cell death by inducing ROS generation.

Oxidative stress is considered to be a major factor contributing to pathogenesis and progression of many diseases. A novel andrographolide-lipoic acid conjugate (AL-1) could protect pancreatic ß-cells from reactive oxygen species (ROS)-induced oxidative injury. However, its protective mechanism is still unclear. In this work, we used proteomics to identify AL-1-regulated proteins in ß-cells and found that 13 of the 71 proteins regulated by AL-1 were closely associated with antioxidation. These differential proteins were mainly involved in the ERK1/2 and AKT1 signaling pathways. Functional investigation demonstrated that AL-1 exerted its protective effects on H2O2-induced cell death of ß-cells by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways. As a consequence, the expressions of antioxidant proteins including Trx1, Prx1 and Prx5, and anti-apoptotic proteins including PDCD6IP, prohibitin, galectin-1 and HSP were upregulated. AL-1 probably worked as a "vaccinum" to activate the cellular antioxidant system by inducing the generation of low concentration ROS which then reciprocally protected ß-cells from oxidative damage caused by high-level ROS from H2O2. To the best of our knowledge, this is the first comprehensive proteomic analysis illustrating a novel molecular mechanism for the protective effects of antioxidants on ß-cells from H2O2-induced cell death.

Identification of novel signaling components in genistein-regulated signaling pathways by quantitative phosphoproteomics.

Isolated from soybeans, genistein is an isoflavonoid that exhibits anti-carcinogenic effects. Genistein could induce G2/M arrest and apoptosis of various cancer cells in vivo and in vitro. Although ERK1/2, AKT, p90RSK and NFκB were previously found to be regulated by genistein, most of signaling components in genistein-inhibited signaling pathways were still unknown. Here, we used SILAC quantitative phosphoproteomics to globally identify the phosphoproteins and their regulatory sites in signaling pathways mediated by genistein. We detected 1177 phosphorylation sites on 635 unique proteins; among them, 320 phosphorylation sites on 222 unique phosphopeptides representing 215 non-redundant proteins were modulated at least 1.5-folds by genistein. Apart from ERK1/2, PI3K, p90RSK, Bad and topoisomerase that are known genistein-regulated effectors, many novel phosphoproteins were identified for the first time to be involved in genistein-regulated signal transduction networks. They mainly include 9 receptors, 5 signal adaptors, 13 protein kinases, 2 protein phosphatase regulatory subunits, and 14 transcription regulators. Several of these phosphoproteins have been proven to be involved in G2/M arrest or apoptosis such as GPCRs, DCC, NCK1, TNK2, BTK, TP53BP1, BCLAF, MAX and MAG. This dataset provides valuable insights into the cancer-related phosphorylation signaling pathways regulated by genistein.

Proteomics characterization of gastrokine 1-induced growth inhibition of gastric cancer cells.

We previously used proteomics technology to globally identify gastric cancer-associated proteins and found that gastrokine 1 (GKN1) was dramatically underexpressed in gastric cancer tissues. Here, we further showed that GKN1 could inhibit cell growth and induce cell cycle arrest in gastric cancer cells. The activity of protein kinase PKCδ/θ was inhibited by GKN1, whereas the activity of ERK1/2 and JNK1/2 was increased by GKN1, suggesting that GKN1 induced growth inhibition of gastric cancer cells by synergistically regulating the activity of these protein kinases. Seventy-four proteins were found to be regulated by GKN1 by proteomics analysis, including α-enolase (ENO1) and Cathepsin D. Interestingly, ENO1 is an important hub in the protein-protein interaction network of the 74 differential proteins. Silencing of ENO1 resulted in growth inhibition and cell cycle arrest of gastric cancer cells, similar to the effect of GKN1 overexpression in cells, whereas ENO1 overexpression blocked GKN1-induced growth inhibition and cell cycle arrest. These observations suggested that ENO1 downregulation played an important role in GKN1-induced growth inhibition of gastric cancer cells.

Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics.

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.