GAP161 targets and downregulates G3BP to suppress cell growth and potentiate cisplaitin‐mediated cytotoxicity to colon carcinoma HCT116 cells

Abstract

Ras-GTPase-activating protein SH3 domain-binding proteins (G3BP) are overexpressed in various human tumors and participate in several signaling pathways involved in growth, differentiation and apoptosis. G3BP interact with RasGAP (Ras-GTPase activating protein) only in growing cells and depend on Ras activation, and participate in the Ras signal pathway. Therefore, the blockage and downregulation of G3BP may be a new strategy for cancer therapy. In this report, we demonstrate that a novel peptide GAP161 blocked the functions of G3BP and markedly suppressed HCT116 cell growth through the induction of apoptosis. The peptide bound with G3BP, which interfered with the interaction of G3BP1 with RasGAP and further suppressed Ras signaling pathways. GAP161 downregulated G3BP1 and G3BP2 proteins. Similarly, the knockdown of G3BP substantially decreased the proliferation of HCT116 cells and inhibited Ras signal pathways. Furthermore, the downregulation of G3BP could enhance cisplatin-induced apoptosis and growth inhibition of HCT116 cells. We also found that GAP161 suppressed the growth of BALB/c mice bearing colon CT26 tumors and nude mice bearing HCT116 xenografts. These results suggest that downregulation of G3BP might be useful in cancer therapy and that GAP161 is a promising new therapeutic agent for cancers. (Cancer Sci 2012; 103: 1848-1856 T he G3BP protein family contains G3BP1 and G3BP2, with similar molecular structures. Both proteins are dramatically overexpressed in human cancers, especially in breast, head and neck, colon and lung.(1-3) G3BP are involved in a variety of growth-related signaling pathways that are involved in carcinogenesis and metastasis, (4,5) including NF-jB, Ras signaling and the ubiquitin proteasome system.(1,6-8) The expression level of G3BP1 changes reciprocally to the level of phosphatase and tensin homolog deleted on chromosome ten (PTEN). The negative effect of PTEN expression on the protein level of G3BP1 is recapitulated by direct inhibition of PI3K.(9) G3BP1 and G3BP2 also interact with p53 and MDM2 to modulate their expression and localization.(10) In addition, G3BP1 is an important marker of stress granules (SG). The NTF2-like domain of G3BP1 mediates its recruitment to SG influenced by Ras. (11) Stresses like hypoxia, heat shock and chemotherapy could induce SG formation, and SG could inhibit apoptosis by suppressing stress-responsive MAPK pathways.(12) Taken together, several lines of evidence suggest that the overexpressed G3BP in a wide range of cancers could be a target for anticancer therapy.It is believed that RasGAP, a regulator of Ras and Rho GTPbinding proteins, (13,14) have dual functions: the negative effect of GTPase pathways and downstream signaling transduction using effectors such as Raf-1, PI3K and MAPK.(15) The SH3 domain of RasGAP is contributed to binding of G3BP and is essential for Ras downstream signaling. Two independent groups found that p120-RasGAP was cleaved by caspases, which induced both anti-apoptotic and pro-apoptotic signals, depending on the ex...

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

“…Interestingly, our observation of similar effects of TP and TP10 on cell viability and cell cycle progression is puzzling since TP was shown to strongly down-regulate GTPase activity followed by decreased cell viability, due to the presence of full-mastoparan sequence, whereas TP10 had no effect on this enzyme [8]. Our findings could be explained by the reported up -regulation of KRAS protein (protein with GTPase activity) in HT29 cells [37] and Ras-GTP-activating protein in HCT116 cells [38] which may overcome the GTPase inhibitory effect of TP. On the other hand, the observed higher cytotoxic effect of TP10-biot1 as compared to TP-10 during prolonged incubation (up to 48 h) with HT29 and HCT116 cells might be caused by increased pore formation in plasma membrane and induction of apoptosis [39].…”

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway

“…However, it has also been suggested that EGCG exerts its anti-oxidative and inflammatory effects through markedly different signaling mechanisms in different disease models and/or cell types. While G3BP1 appears to be the direct target in lung cancer cells [19] , our results indicate that this is not the case in human LX-2 cells because the knockdown or overexpression of G3BP1 did not alter the EGCG-mediated repression of COL1A1, TGF-β1, or MMP-2 expression (Figure 4). Instead, our findings indicate that EGCG exerts its anti-fibrotic and inflammatory effects through the PI3K/Akt signaling pathway in both LX-2 cells and the rat liver (Figure 1-3, 5), in agreement with the findings of Xiao et al [9] Most importantly, the lack of an additive effect of EGCG and LY294002 (a specific inhibitor of PI3K) on the repression of the TGF-β1-stimulated phosphorylation of Smad2 and Nevertheless, verification of the EGCG-mediated repression of TGF-β1-stimulated COL1A1 expression in LX-2 cells suggests that the findings involving the BDL rats in the current study and those involving CCl 4 -treated rats [7] and high-fat-dietfed rats [9] from previous reports have clinical implications in human liver fibrosis.…”

“…As shown in Figure 4A, TGF-β1 markedly stimulated G3BP1 protein expression in the LX-2 cells, and the addition of EGCG substantially repressed this stimulation. To gain further insight into the role of G3BP1 in the antifibrotic effects of EGCG, we altered G3BP1 expression in LX-2 cells by siRNA knockdown or by over-expression using a plasmid construct [19] . As shown in Figure 4B and 4C, neither the reduction nor over-expression of G3BP1 altered the EGCG-mediated repression of the mRNA expression of COL1A1, MMP-2, SMAD4, and TGF-β1 after TGF-β1 stimulation.…”

MicroRNA-23b Targets Ras GTPase-Activating Protein SH3 Domain-Binding Protein 2 to Alleviate Fibrosis and Albuminuria in Diabetic Nephropathy

“…3 G3BP2 acts through NF-kB, Ras signaling, and the mitogenactivated protein kinase pathways. 4 Within the mitogen-activated protein kinase family, p38 kinase (p38) and c-Jun N-terminal kinase are activated in response to hyperglycemia. Increased activation of p38 has been identified as a fundamental mechanism responsible for renal dysfunction in diabetes.…”

“…4,38,39 Liu et al reported that mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNAdependent manner. 40 In this study, we have confirmed that downregulation of G3BP2 by miR-23b could decrease kidney fibrosis and alleviate proteinuria in DN.…”