25-hydroxycholesterol–induced cell death via activation of ROCK/LIMK/cofilin axis in colorectal cancer cell spheroids

•25OHC induces cofilin phosphorylation in colorectal cancer spheroids.•Cytochalasin D and a LIMK inhibitor inhibit 25OHC-induced cell death in spheroids.•25OHC activates the ROCK pathway and actin rearrangement via caspase-3 activation.•DLD1 and HT29/WiDr cells differ in drug responsiveness and spheroid structure. 25-Hydroxycholesterol (25OHC) induces anchorage-dependent programmed cell death, or anoikis, in colorectal cancer cells but the mechanism is not fully understood. Here, we found that 25OHC induced cofilin phosphorylation and promoted rearrangement of the actin cytoskeleton in spheroids of the colorectal cancer cell lines, DLD1 and HT29/WiDr. Cell death induced by 25OHC was inhibited by the actin polymerization inhibitor, cytochalasin D, and BMS-3, an inhibitor of LIMK, which phosphorylates and inactivates cofilin. In addition, we showed that cofilin phosphorylation induced by 25OHC was associated with caspase-3 activation, which can activate ROCK. Rho GTPase was directly activated by 25OHC. These results indicate that 25OHC affects actin dynamics through activation of the Rho/ROCK/LIMK/cofilin axis, eventuating in the cell death of colorectal cancer cell spheroids.