Abstract Sodium butyrate (SB) -C-4 saturated fatty acid (short-chain fatty acids) present in the human bowel membrane in the quite high concentration (2 mM) as food metabolites. It was previously reported that SB showed anti-tumor effect as HDAC inhibitor, however, the precise mechanism has not been elucidated yet. Here, we focused on the role of SB on cancer cell growth, motility and invasion. Physiological concentration of SB (0.25-4 mM) induced inhibition of cell growth (2D), colony formation in soft agar, in vitro motility and invasiveness with Boyden chamber and wound healing assay in a dose-dependent fashion using human fibrosarcoma (HT1080), glioblastoma (A172) cells and rat mammary Walker 256 cells. SB also affected the morphology of HT1080, A172 and Walker 256 cells, namely spread out with marked stress fibers bridging focal adhesions. Phosphorylation levels of focal adhesion kinase (Y577 and Y397 sites) were increased but that of myosin light chain (S19 site) was not altered. All of these biological effects of SB were reversible, and recovered after withdrawal. In contrast, HDAC inhibitor trichostatinA inhibited cancer cell growth irreversibly but not affected motility, invasiveness and morphology. In addition, HT1080 and A172 cells treated with SB showed positivity for senescence-associated β-gal (SA-β gal) staining with elevated expression levels of p53 and p21 proteins in a time and dose-dependent manner. In contrast, mRNA level of p21 was sharply declined after the treatment with SB. Knocking down of p21 protein expression by siRNA reversed both the growth inhibition and positivity for SA-β gal staining but not the inhibition of invasiveness. Thus p21 was required for cellular senescence but not for the decrease in motility induced by SB. Further, proteasome inhibitor, MG132 decreased the cellular growth and inhibited motility and invasiveness of the HT1080 and A172 cells, and showed similar all these biological effects of SB. Finally, intrathecal administration of SB effectively inhibited the invasion of Walker256 cells into the brain and elongated the animal (rat) survival in vivo. Collectively, SB inhibited growth with induced cellular senescence, inhibited invasion by distinct mechanism, and thus would be a good candidate for anti-invasive therapy without severe adverse effects. Citation Format: Kazuyuki Itoh, Kiyoko Yoshioka, Hidemitsu Nakagawa. Sodium butyrate induced cellular senescence and inhibited invasion of cancer cells with distinct mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2624. doi:10.1158/1538-7445.AM2013-2624