Shin-Jung Choi

RhoB induces apoptosis via direct interaction with TNFAIP1 in HeLa cells.

RhoB, a tumor suppressor, has emerged as an interesting cancer target, and extensive studies aimed at understanding its role in apoptosis have been performed. In our study, we investigated the involvement of RhoB‐interacting molecules in apoptosis. To identify RhoB‐interacting proteins, we performed yeast‐two hybrid screening assays using RhoB as a bait and isolated TNFAIP1, a TNFα‐induced protein containing the BTB/POZ domain. The interaction between RhoB and TNFAIP1 was demonstrated in vivo through coimmunoprecipitation studies and in vitro binding assays. RFP‐TNFAIP1 was found to be partially colocalized with EGFP‐RhoB. The partial colocalization of RhoB and TNFAIP1 in endosomes suggests that RhoB‐TNFAIP1 interactions may have a functional role in apoptosis. TNFAIP1 elicited proapoptotic activity, while simultaneous expression of RhoB and TNFAIP1 resulted in a dramatic increase in apoptosis in HeLa cells. Furthermore, knockdown of RhoB using siRNA clearly rescued cells from apoptosis induced by TNFAIP1. This finding suggests that interactions between RhoB and TNFAIP1 are crucial for induction of apoptosis in HeLa cells. The observation of increased SAPK/JNK phosphorylation in apoptotic cells and the finding that a JNK inhibitor suppressed apoptosis indicates that SAPK/JNK signaling may be involved in apoptosis induced by RhoB‐TNFAIP1 interactions. In conclusion, we found that RhoB interacts with TNFAIP1 to regulate apoptosis via a SAPK/JNK‐mediated signal transduction mechanism.

NSC126188, a piperazine alkyl derivative, induces apoptosis via upregulation of RhoB in HeLa cells

SummaryWe describe here a piperazine alkyl derivative, NSC126188, which induced apoptosis of HeLa cells by upregulating RhoB expression. NSC126188 caused multi-septation of fission yeast and hypersensitized a ∆rho3 mutant, which implicates the involvement of functional human homolog RhoB. The treatment of cells with NSC126188 induced apoptosis and a dramatic increase in RhoB expression. In addition, RhoB knockdown using siRNA rescued cells from apoptosis, indicating a crucial role of RhoB in NSC126188-induced apoptosis. In a reporter assay using luciferase and EGFP under control of the RhoB promoter, NSC126188 increased both luciferase activity and the expression of EGFP, implicating transcriptional activation of RhoB by NSC126188. Furthermore, NSC126188 demonstrated in vivo anti-tumor activity, inhibiting tumor growth by 66.8% in a nude mouse xenograft using PC-3 human prostate cancer cells. These results suggest that NSC126188 is a potential lead compound and that upregulation of RhoB is associated with NSC126188-induced apoptosis.

Identification of small molecules inducing apoptosis by cell-based assay using fission yeast deletion mutants.

SummaryThe cell-based assay using yeast deletion mutants has been recognized as an efficient analysis to discover therapeutic compounds and reveal their mode of action. In this study, S. pombe deletion mutants-based HTS screening was carried out to identify potential anti-cancer agents. The NCI chemical library of 5700 compounds was screened using kit strains, which consisted of S. pombe mutants harboring deletions in genes involved in DNA repair and mitotic control. During the screening, we identified 40 compounds conferring growth inhibition of S. pombe. Their anti-tumorigenic properties were examined by phenotypic effect on S. pombe, flow cytometry and apoptosis analysis of human cancer. Here, we report hit compounds inducing apoptosis for development of anti-cancer agents suggesting that S. pombe deletion mutants are useful in identifying potential anti-cancer agents in human cancer therapeutics.

Rapid screen of human genes for relevance to cancer using fission yeast

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis. (Journal of Biomolecular Screening 2007:568-577)