RU486 Disturbs The Microfilament Cytoskeleton Reorganization Of MCF-7 Breast Cancer Cells By Inhibiting RhoA/ROCK Signal Pathway

RU486,the progesterone receptor blocker,also has anti-tumor effects,one of which is the inhibiting of cell migration,as well as changing cell morphology.It is known that the morphology and migration ability of tumor cells are closely related with microfilament skeleton reorganization.However,the relationships between RU486 and the microfilament cytoskeleton still lack an experimental demonstration.In this study,human breast cancer MCF-7 cells were utilized to investigate how filamentous actin(F-actin),a microfilament protein,changes under an RU486 effect,and if this phenomenon results from the Rho A/ROCK signal pathway,thereby providing new clues for the anti-tumor mechanism study of RU486.Methods 1.The expression and distribution of F-actin in MCF-7 cells treated with different concentrations of RU486 were observed by fluorescence staining.2.The protein levels of Rho A,ROCK1,ROCK2 and F-actin in HBL-100,MCF-7 and MDA-MB-231 cells were detected through the Western blot.3.Effects of RU486 on the Rho A/ROCK signal pathway.(1)Groups.The control group was treated with a DMSO solvent,while the five experimental groups were treated with the following drugs:(1)Rho A/ROCK signaling activator Narciclasine(NAR).(2)Rho A/ROCK signaling inhibitor GSK429286A(GSK).(3)RU486(RU).(4)Narciclasine combined with RU486(NAR+RU).(5)GSK429286A combined with RU486(GSK+RU).(2)The morphology and distribution of F-actin were observed by fluorescence staining.(3)A scaling experiment as well as Transwell chamber experiments were performed,so as to detect the ability for cell migration.(4)The expression levels of Rho A,ROCK1,ROCK2,p LIMK,p MYPT and F-actin were identified by Western blot.4.All of the experimental results were carefully analyzed by utilizing SPSS software.Furthermore,all the data were expressed as "mean ± standard ",while the t test was employed when two groups of data were compared.The ANOVA analysis was utilized when three or more groups of data were examined,while P<0.05 was considered as statistically significant.Results 1.RU486 alters the arrangement and distribution of the MCF-7 microfilament skeleton F-actin.The results of the fluorescence staining illustrated that the cell morphology and F-actin distribution was altered after being treated with RU486.Along with the increasing RU486 concentration,the cell shape gradually became polarized,the cell body and nucleus became elongated,and the F-actin on the cell membrane did not exhibit a continuous fluorescent expression,but rather,an uneven thickness.Dense fluorescent lumps appeared in the cell poles or corner protuberances,while the intracellular F-actin structure remained sparse,and under fluorescent light,appeared condensed into thick,massive lines.2.The expression of Rho A,ROCK,and the F-actin protein in breast cancer cells appeared higher than that of normal breast epithelial cells.The results of the Western blot illustrated that Rho A,ROCK1,ROCK2 and F-actin were detected in both a normal breast epithelial HBL-100 cell line and breastcancer MCF-7 or MDA-MB-231 cell lines.However,the protein levels were higher in two kinds of breast cancer(P<0.05 vs HBL-100).3.The effects of RU486 changing the F-actin distribution and arrangement in MCF-7 cell are closely related to the Rho A/ROCK signaling pathway The fluorescence staining revealed that the cell body spread and that the cytoplasmic fibroin F-actin fibers were thickened,especially around the nucleus,after the activator treatment alone.Also,there were more pseudo-pods on the membrane.When combined with RU486,the pseudo-like structure and the grid-like stress fiber reduced.After the inhibitor treatment,however,the F-actin fibers were sparse,broken or an agglomerated lumpy fluorescence.when combined with RU486,the stress fiber distributing more uneven density,and there were small wrinkled protrusions visible on the membrane.4.The effects of RU486 inhibiting MCF-7 cell migration are related to the Rho A/ROCK signaling pathway The results of the scratch test revealed that the cell mobility increased after the activator treatment alone(P<0.05 vs DMSO),however,was reduced after the inhibitor or RU486 alone treatment(P<0.05 vs DMSO).The cell mobility of the activator combined with RU486 treatment was less than that of NAR(P<0.05 vs NAR),but much more accentuated than when RU486 treatment was administered(P<0.05 vs RU)alone.Moreover,the cell mobility of the inhibitor combined with RU486 treatment was less than that of the inhibitor or RU486(P<0.05 vs GSK or RU)alone.The transwell migration assay indicated that the number of migrating cells increased after the activator treatment alone(P<0.05 vs DMSO),but was reduced after the inhibitor or RU486 alone treatment(P<0.05 vs DMSO).The number of the migrating cells with the activator when combined with RU486 treatment was less than that of NAR(P<0.05 vs NAR),but much more so than RU486 treat alone(P<0.05 vs RU).Furthermore,the number of migrating cells with the inhibitor combined with RU486 treatment was less than that of the inhibitor or RU486(P<0.05 vs GSK or RU)alone.5.RU486 reduced the level of the Rho A/ROCK signaling pathway in MCF-7 cells The results of the Western blot indicated that Rho A,ROCK1 and ROCK2 protein levels decreased with the increase of drug concentration(P<0.05 vs DMSO),while the lowest protein level was at 30 ??(P <0.05 vs 20 ?M).The F-actin protein levels were statistically significant at 30 ?M(P< 0.05 vs 0 ?M)only.After 20 ?M RU486 was applied to the cells at diverse intervals,the protein levels decreased along with time(P<0.05 vs 0 h),and reached the lowest levels at 72 h(P<0.05 vs 48 h).When compared among different time points,the level of Rho A and ROCK protein decreased with the prolongation of time at 24 h,48 h and 72 h(P<0.05 vs 0 h)after 20 ?M RU486 treated,and the protein level was the lowest at 72 h(P<0.05 vs 48 h).The F-actin protein level decreased at 48 h(P<0.05 vs 0 h),the lowest at 72 h(P<0.05 vs 48 h).When treated with the activator alone,the p MYPT and p LIMK protein levels were gradually up-regulated(P<0.05 vs DMSO)in a period of 24 h.While using the inhibitor or RU486 alone,these phosphorylated protein levels went down(P<0.05 vs DMSO).When RU486 combined with the activator,these phosphorylated protein levels were less than activator alone(P<0.05 vs NAR),but higher than that of RU486 alone(P<0.05 vs RU486).When RU486 combined with the inhibitor,these phosphorylated protein levels were lower than when using both of the two respectively(P<0.05 vs GSK or RU).Conclusions 1.RU486 interferes with the microfilament skeleton reorganization of the breast cancer MCF-7 cell,and then alters the cell morphology,thereby affecting migration.2.The mechanism of the RU486 effects is closely correlated to the Rho A/ROCK signal pathway.