Cell migration is a prerequisite for
cancer invasion and
metastasis, suggesting cell motility as a potential therapeutic target for
cancer treatment. A synthetic library was screened to identify inhibitors of
tumor cell migration. From this, we discovered that CAC-1098 (
aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized
KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the
bromide group of CBI-0997 with a methoxyl group. Like CBI-0997,
KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because
KRIBB3 has a better
drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding
proteins. Using affinity chromatography, we identified Hsp27 as a target
protein of
KRIBB3 in vitro. Treatment of MDA-MB-231 cells with
phorbol 12-myristate 13-acetate induced
protein kinase C-dependent phosphorylation of Hsp27 and
tumor cell migration. In contrast, treatment of MDA-MB-231 cells with
KRIBB3 blocked
phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and
tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of
KRIBB3 on
tumor cell invasion, and knockdown of Hsp27 using
small interfering RNA inhibited
tumor cell migration. Overall, our results demonstrate that
KRIBB3 inhibits
tumor cell migration and invasion by blocking
protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.