Myosin Light Chain Kinase Inhibitor Induces Apoptosis In Vitro And In Vivo

Our data demonstrate that MLC are dephosphorylated when cells are treated with a variety of agents that cause apoptosis. Of the apoptotic agents that we tested, only dexamethasone did not cause a decrease in MLC phosphorylation. Although dexamethasone induces apoptosis in a number of cell types, it did not cause smooth muscle cells to die, further supporting the idea that MLC dephosphorylation and apoptosis are connected. MLC are also dephosphorylated when the cytoskeleton was disrupted with cytochalasin D and by inducing anoikis. The implication of these data is that MLC dephosphorylation is involved in apoptosis as induced by very different mechanisms.2． Inhibition of MLCK decreases MLC phosphrylation and induces apoptosisEqually important is the observation that inhibiting MLCK and decreasing MLC phosphorylation induces apoptosis. Two separate approaches were used to establish the importance MLC dephosphorylation in apoptosis. First, we showed that ML-7, a relatively specific inhibitor of MLCK. induced MLC dephosphorylation and stimulated apoptosis in SMC. We have also found that another MLCK inhibitor, KT 5926, also induces apoptosis (not shown). As a more direct approach, we microinjected an affinity-purified antibody that specifically inhibits MLCK into cells. This approach also resulted in apoptosis. Thus, MLC dephosphorylation secondary to MLCK inhibition appears to be sufficient to induce apoptosis.3．The Phosphorylation level of MLC during apoptosis and commitment in apoptosisTime course studies on cells treated with actinomycin D show an initial increase followed by a decrease in MLC-P. This initial increase in MLC-P is consistent with previous reports that have implicated a role for increased MLC-P in membrane blebbing, an early event in apoptosis. Thus, there appear to be 2 phases of the MLC-P response during apoptosis: and correlates with blebbing and a second phase that involves MLC-DP. The second phase correlates with the commitment to apoptosis and is associated with caspase activation and the commitment to cell death. 4． Apoptotic signal transduction during apoptosis induced by ML-7The demonstration that inhibiting MLCK is sufficient to induce cell death suggests that MLC-DP may be part of a broader mechanism involved in determining cell fate. The properties of the cytoskeleton are determined mainly by filaments composed of actin and myosin II. Interestingly, inhibiting MLCK, cytochalasin D and loss of cell contacts share in common the translocation of Bmf to mitochondria and caspase activation. The translocation of Bmf to the mitochondria is reported to antagonize Bcl-2 and result in caspase activation. Consistent with these observations, we found in rescue experiments that overexpressing Bcl-2 or inhibiting caspases prevented SMC apoptosis. In summary, the data presented above demonstrate that myosin II is dephosphorylated during apoptosis and that inhibiting MLCK is sufficient to induce apoptosis. The in vitro data also demonstrate that apoptosis induced by MLC-DP, cytochalasin D and anoikis apparently share a common pathway that involves the destabilization of the cytoskeleton. 5． MLCK inhibitor ML-7 induces cancer cells apoptosis in VIVOInterestingly, the in vivo data demonstrate that inhibiting MLCK induces apoptosis in breast cancer cells and retards the growth of tumor in mice. Tumor volume and weight are smaller in ML-7 group than that of control group. TUNEL staining of tumor tissue shows the TUNEL positive cells percentage is much higher in ML-7 treated mice tumor than that of control group.6． The synergic antitumor effect of ML-7 on other anti-cancer regeantIn Vitro experiment showed the annexin V positive cell percentage of combined ML-7 with 100mM etoposide is as high as that in 1000mM etoposide alone group in both Mat-ly-lu cells and Mm5MT cells. The inhibiting tumor rate of ML-7 combined with etoposide group is much higher than either ML-7 treated alone or etoposide treated alone group in both mice b...