Mode of action of a novel piperazine alkyl anti-cancer agent KHG2 regulating RhoB expression and functional study of RhoB increasing apoptosis of cancer cells

Abstract

RhoB plays a role as a tumor suppressor, causing growth-inhibitory and apoptotic effects on cancer cells and also known to be a critical mediator for the apoptosis by many anti-cancer agents. Here, we report a novel piperazine alkyl derivative, KHG2 for an anti-cancer drug, which upregulate RhoB expression, leading to apoptosis in human cancer cells. KHG2 showed a good cytotoxicity toward human cancer cells in-vitro and mouse xenograft assay using NUGC-3, a human stomach cancer cell line. KHG2 induced a G2-M arrest and apoptosis in NUGC-3. KHG2 activated JNK pathway, thereby increasing phospho-c-jun in NUGC-3. And, prior treatment of a JNK inhibitor, SP600125, inhibited most of the apoptosis, suggesting that the activation of JNK might play a crucial role for the apoptosis by KHG2. Furthermore, whereas KHG2 decreased phospho-forms of both PTEN (Ser380) and Akt (Thr308) as well as total amounts of FTase-1α and K-ras, it dramatically increased p-Bcl2 (Ser70) and RhoB. The functional role of RhoB was proved by ectopic expression of the gene in NUGC-3 that induced the eminant apoptosis. These results clearly show that a novel compound, KHG2, induces the anti-cancer activity via dysregulation of the multiple pathways including activation of JNK and RhoB, thereby inducing apoptosis in NUGC-3. In spite of the many previous reports of anti-cancer activities of RhoB, the exact mechanisms for the apoptosis by RhoB are not proved well. Here, we report that TNFAIP1 induces an apoptosis through direct interaction with RhoB in Hela, a human cervical cancer cell line. Through the yeast-two hybrid screening using Hela cDNA library as a prey, we found many interesting proteins including TNFAIP1. RhoB also interacted with TNFAIP1 in-vitro and In-vivo, Furthermore, co-transfection of both RhoB and TNFAIP1 induced eminent apoptosis compared to single transfection. And RhoB knock down using RhoB-specific si-RNAs inhibited the apoptosis by TNFAIP1 in Hela cells, suggesting tha...