Abstract
PURPOSE: Recently, we found that erufosine (erucylphospho-N,N,N trimethylpropylammonium) can induce up-regulation of RhoB expression in oral squamous carcinoma (OSCC) cells, thereby hinting at a tumor suppressive role. Therefore, we aimed to evaluate the role of RhoB in the tumor suppressive mode of action of erufosine on OSCC cells. METHODS: Anti-proliferative effects of erufosine were determined in HN-5 and FaDu OSCC-derived cells using a MTT assay. RhoB up-regulation was detected using microarray and qRT-PCR-based expression assays at IC(25), IC(50) and IC(75) concentrations of erufosine. The results obtained were verified by Western blotting. In addition, siRNA-mediated RhoB knockdown was carried out and combined with erufosine treatment, after which cell cycle, colony formation and migration assays were performed to evaluate its combined effects. RESULTS: We found that after erufosine treatment of HN-5 and FaDu cells for 24, 48 and 72 h the IC(50) values ranged from 43 to 37 μM and 27- to 15 μM, respectively. Microarray and qRT-PCR-based expression analyses revealed RhoB up-regulation up to 9-fold and 20-fold, respectively. Using Western blotting, an increase in RhoB protein expression was observed, as well as a decrease in pAkt (Ser(473) and Thr(308)) expression and an increase in PARP cleavage. Combined siRNA-mediated RhoB knockdown and erufosine treatment resulted in slightly reduced RhoB and pAkt levels compared to erufosine treatment alone. Subsequent cell cycle analyses revealed an increased apoptotic induction, but a reduced G2 cell cycle arrest, of the combination. At the functional level, synergistic effects were observed using cell migration and colony formation assays. CONCLUSIONS: Our data show that erufosine can cause up-regulation of RhoB expression in OSCC cells. Combining erufosine treatment with siRNA-mediated RhoB knockdown did, however, not reveal a role of RhoB in its tumor suppressive mode of action.