The Mechanisms Of Proliferation, Morphology And Adhesion Control In Human Ovarian Cancer Cell Lines HO-8910 By Glucocorticoids

Glucocorticoids (GCs) play an important role in regulating cell gowth, differentiation, apoptosis and adhesion in many cell types. Although it has been known that the biological effects are predominantly mediated by glucocorticoid receptor (GR), a ligand-dependent transcriptional factor, through regulating the expression of its target genes, few target genes have been identified to mediate GCs biological effect. Our previous studies demonstrated that Dex markedly induced the growth arrest and pronounced morphologic alteration in HO-8910 cells. Moreover, Dex treatment could prolong the detachment time when cells were digested with trypsin, suggesting that Dex increases the adhesion ability of HO-8910 cells to extracellular matrix (ECM). The purpose of this paper is to explore the possible mechanisms of proliferation, morphology and adhesion control in human ovarian cancer HO-8910 cells by glucocorticoids.In the first part of the paper, we studied the role of small G protein RhoB in Dex-induced growth arrest to know the molecular mechanisms responsible for the anti-proliferative effect of GCs.Rho proteins, monomeric GTPases of the Ras superfamily, are well known to regulate cell adhesion, motility, proliferation and survival. It is demonstrated that RhoB, as a member of Rho subfamily, plays a tumor-suppressive role in several human cancer cells. So we examined whether the expression of RhoB could be regulated by Dex, and then explored the possible role of RhoB in Dex-induced anti-proliferation and morphological alteration. This study also will help to realize the mechanism of the transformation inhibition by Dex in normal ovary epithelial cells.The expressions of Rho proteins regulated by Dex in mRNA and protein levels in HO-8910 cells were examined firstly by semi-quantitative RT-PCR and Western blot, respectively. We found that Dex could induce the expressions of small GTPase RhoB mRNA and protein, but not RhoA and RhoC mRNA in a dose- and time- dependent fashion via GR in HO-8910 cells. And the induced-expression of RhoB by Dex can beobserved in several other human tumor cells. To know whether the regulation of RhoB expression by Dex occurs in the transcription level, we transiently transfected the HO-8910 cells with a luciferase reporter construct containing a ~1.9 kb (-1756/+111) fragment of the promoter sequence of human RhoB gene and found that the activity of the RhoB reporter gene did not increase obviously. This at least indicated that there is no functional GRE in this promoter region. This result can not exclude the possibility that Dex may regulate RhoB expression through interfering the function of other transcriptional factor such as AP-1 or NF-kB, or through post-transcriptional level such as increasing the stability of RhoB mRNA.The role of RhoB in Dex-induced growth arrest in HO-8910 cells was further determined by transfection method. The result from MTT assay and soft agar assay showed that over-expression of RhoB increased Dex-induced growth inhibition, while inhibition of RhoB expression by RNA interference reversed the Dex-induced growth arrest in HO-8910 cells, indicating that RhoB signaling is involved in the Dex-induced proliferation inhibition in these cells. We also found that ectopic expression of RhoB could induce the cell shape change similar to the case with Dex treatment in HO-8910 cells, while inhibition RhoB expression showed no notable change in cell shape, indicating that RhoB signaling does not play important role in Dex-induced morphological alteration.Known as a downstream effector of Rho GTPases, Rho associated kinase (ROCK) is an important regulator of actin cytoskeleton reorganization. So we want to know whether ROCK may play a regulatory role in Dex-induced morphological change. Western blot analysis showed that Dex could up-regulate the expression of the type II ROCK protein (ROCK II) in a dose- and time- dependent manner. Whereas, inhibiting ROCK activity by Y-27632 could not reverse Dex-induced morphologic alteration, indicating that ROCK may not be responsible for this process.There is growing evidence that transcription factor NF-κB is implicated in the control of cell cycle progression and tumor formation by promoting proliferation and inhibiting apoptosis. We studied the possible effect of RhoB on NF-κB activity in HO-8910 cells by reporter gene assay in order to clarify the functional connection ofRhoB signaling and NF-κB activity. The result showed that over-expression or activation of RhoB signaling elevated the basal transcriptional activity of the transcription factor NF-κB. Furthermore, elevating RhoB signaling enhanced the inhibitory effect of Dex on NF-κB activity, while attenuating RhoB signaling almost abrogated Dex suppression of NF-κB signaling, indicating that the RhoB pathway is involved in the regulation of NF-κB activity and is essential for Dex transcriptional repression on NF-kB signaling in HO-8910 cells.To the cells growing in the adherence manner, attachment of cells to the ECM is one of the preconditions to growth and differentiation. Clarifying the mechanism of Dex-promoted adhesion ability may help to know the pathway through which Dex plays anti-apoptosis effect in HO-8910 cells. So, in the second part of the paper, we explored the possible mechanisms of the Dex-induced adhesion ability in HO-8910 cells through the aspects of ECM and integrin β1 who have vital influence on the cell adhesion process.The effect of Dex on the HO-8910 cells adhesion to the ECM was firstly demonstrated by cell adhesion assay. The results showed that Dex indeed enhanced the cell adhesion ability to one of the ECM component, fibronectin (FN), in a time-dependent manner without changing the expression of FN mRNA. Of course, the result does not exclude the possibility that Dex may facilitate the expression and excretion of FN protein and other ECM component.We further examined the expression of integrin β1 mRNA and protein by semi-quantitative RT-PCR and Western blot, respectively. It is showed that Dex could induce the expression of integrin β 1 protein but not mRNA in a time- and dose- dependent manner. The increase of the cell-surface expression of integrin β1 protein was also demonstrated by immuno-fluorescent flow cytometry analysis. This indicated that Dex could enhance the function of integrin β 1 through facilitating protein expression and translocation to membrane. We also found that inhibiting the ability of integrin β1 protien by it's blocking antibody could partially reverse the effect of Dex on cell adhesion, indicating that integrin β1 protein mediates the enhancement of adhesion ability to FN by Dex. Since the integrins exert their effect in heterodimer form, we next examined which integrin a protein participated in the process. Western blot analysis showed that Dex...