To investigate the effect of
blebbistatin (
BLEB, a selective
myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions.
BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM)
myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive
reverse transcriptase PCR. SM
myosin heavy chain (MHC), non-muscle (NM) MHC
isoforms (NMMHC-A and NMMHC-B), and
proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections.
BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of
BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of
BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved
caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream
proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally,
BLEB decreased SM cell number and SM MHC expression, along with attenuated
phenylephrine-induced contraction and altered prostate SMM
isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate
BLEB regulated
myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM
isoform composition.