Cannabidiolic acid causes a defect in tail retraction of migrating MDA-MB-231 cells: Possible involvements of Rho-associated protein kinases (ROCKs) inhibition and accumulation of vinculin at the rear of migrating cells

We previously reported that cannabidiolic acid (CBDA), a major cannabinoid constituent of the fiber-type cannabis plant, abrogates the migration of highly aggressive human breast cancer MDA-MB-231 cells and activates the small GTPase RhoA by inhibiting protein kinase A. However, the mechanism(s) mediating RhoA signaling, which decreases cell migration, have not yet been comprehensively elucidated. RhoA is an upstream mediator of Rho-associated kinases (ROCKs), diaphanous-related formins (DIAPHs), the RhoA-ROCK pathway (tail retraction), and the RhoA-DIAPH pathway (lamellipodia formation). Herein, we identified CBDA as an inhibitor of ROCKs (at approximately 25 μM), which markedly elongated the cell body of MDA-MB-231 cells, similar to Y-27632, an established ROCK inhibitor. CBDA stimulated lamellipodia formation at the leading edge, whereas NSC23766 (an established Rac1 inhibitor) completely blocked this elongated morphology. Biochemical analyses, including time-lapse imaging and confocal laser scanning microscopy, revealed that, compared to Y-27632, CBDA can induce impaired tail retraction coupled with unidirectional elongation of the cell body, upregulate the mRNA expression of DIAPHs, and accumulate vinculin, an adhesion protein, at the trailing edge without affecting its expression. These results indicate the potential of CBDA as a new candidate for the synthesis of ROCK inhibitors, which can evoke the directed elongation of MDA-MB-231 cells.