| Part ⅠRole of ROCK in increased melanoma migration, invasion and metastasis by glucocorticoidBACKGROUDS AND AIMS:Due to the development of industrialization and modernization, rapid pace of life style and fierce competition, more and more population were suffering chronic stress. Currently,pre-clinical and clinical studies demonstrated that the increasing incidence of tumor and much younger tumor patients is closely associated with durable and chronic stress.Glucocorticoid, an important stress hormone, plays a critical role in the processes of immunosuppression, anti-inflammation and homeostasis sustaining, as well as being involved in the processes of embryonic development and traumatic repairment. Synthetic GCs are widely prescribed therapeutics for the treatment of numerous inflammatory disorders and cancers. The level of GCs in the body is increased not only by stress, but also by medication therapy. Therefore, the relationship between GCs and solid tumor progression has been paid an important attention by people. It is very biologically significant to elucidate the roles of GCs in solid tumor progression.Tumor metastasis is the leading factor that contributes to eventual death in tumor patients. Tumor metastasis is a multistep process that involves migration of tumor cells,local invasion, entry into the circulation, arrest at secondary sites, extravasation, and colonization. So far, studies on GCs affecting tumor migration, invasion and metastasis are very limited. These studies revealed different roles of GCs on tumor metastasis. It is unclear whether the inconsistent role of GCs on tumor metastasis is associated with different tumor type.Melanoma is characteristic of multiple metastasis and high mortality in skin cancers.Clinical data have been demonstrated that the expression of GR is associated with melanoma Clark level, indicating that GCs play a role in melanoma progression to some extent. However, the role of glucocorticoid on melanoma progression is still unknown. In this study, we firstly investigated the effect of glucocorticoid on melanoma migration and invasion in vitro and in vivo and its possible mechanism. We focused on the role of ROCKin the increased melanoma migration, invasion and metastasis by glucocorticoid.METHODS:For drug treatment, Dex(a synthetic glucocorticoid, 100 n M) was used to treat melanoma cells. Protein and m RNA expression were measured by Western blot and q RT-PCR. Boyden transwell migration assay, cell scratch assay and invasion assay were used to measure cell migration and invasion. Cell adhesion assay was used to measure cell adhesion to ECM. Cell proliferation was detected by MTT assay. Rho activity was measured by Rho-GTP pull down assay. Secreted Fibronectin was measured by ELISA. In vivo studies, C57BL/6 mice and BABL/c nude mice were injected via tail vein to establish experimental metastasis model. Corticosterone(50 mg/L) in drinking water was given for12 days, then the lung metastasis foci were observed and counted. Black nodules in the lung were convinced by HE staining.RESULTS:1. We found that Dex(100n M), a synthetic glucocorticoid, promoted melanoma B16F10 and A375 cells adhesion to ECM but had no effect on melanoma cell proliferation.Dex significantly promoted melanoma cell migration and invasion. In vivo studies showed that drinking water containing 50mg/L corticosterone for 12 days promoted melanoma B16F10 cells to metastasize to lung tissue in experimental metastasis C57BL/6 and nude mice models.2. We found that Dex(100n M) had no effect on Rho A, Rho B, Rho C protein expression and activity. Dex(100n M) significantly upregulated ROCK1/2 protein expression in a time-dependent manner but had no effect on ROCK1/2 m RNA expression.GR mediated ROCK1/2 upregulation by Dex. The protein stability of ROCK1/2 was increased by Dex. In vitro studies showed that Y-27632(ROCK1/2 inhibitor) and Fasudil(ROCK2 inhibitor) significantly abrogated the pro-migration and pro-invasion effect of Dex in melanoma cells. In vivo studies showed that Y-27632 significantly abrogated the effect of corticosterone promoting B16F10 cells to metastasize to lung tissue. These data demonstrated that upregulation of ROCK1/2 by glucocorticoid contributed to the pro-migration and pro-invasion effect of glucocorticoid in melanoma.3. We found that Dex(100n M) increased the level of p-AKT in B16F10 and A375 cells. Wortmanin, a PI3K/AKT inhibitor, abrogated the pro-migration and pro-invasion effect of Dex in melanoma cells, indicating that activation of PI3K/AKT by Dex also contributed to pro-migration and pro-invasion effect of glucocorticoid in melanoma cells.Modulation of ROCK expression by PI3K/AKT signaling pathway is cell context-dependent.4. Finally, we found that upregulation of Fibronection expression and secretion by Dex partially contributed to pro-adhesion effect of glucocorticoid in melanoma cells,which may promote cell survival, cell infiltration into metastasis foci and growth. Dex had no effect on MMP-2,3,7,9 protein expression but downregulated TIMP-2 protein expression in B16F10 and A375 cells.CONCLUSION:Taken together, we demonstrated that glucocorticoid promoted melanoma migration and invasion in vitro and in vivo mainly by upregulation of ROCK and activation of PI3K/AKT. Activation of PI3K/AKT may be associated with enhanced cell adhesion to ECM by Dex in melanoma cells. Down-regulation of TIMP-2 may be involved in pro-invasion process of Dex in melanoma cells. These findings for the first time provide a deep understanding of roles of glucocorticoid in melanoma progression.Part ⅡRho B regulates the function of macrophages in the hypoxia-induced inflammatory responseBACKGROUNDS AND AIMS:Immune cells, particularly macrophages, play critical roles in the hypoxia-induced inflammatory response. The hypoxic microenvironment appears to alter the biological behavior and function of macrophages by regulating the expression of genes involved in processes such as metabolism, motility, apoptosis, and the secretion of cytokines and chemokines. However, the precise molecular mechanism responsible for the regulation of macrophage function during hypoxia remains elusive. The small GTPase Rho B is usually rapidly induced by a variety of stimuli and has been described as an important regulator of cytoskeletal organization and vesicle and membrane receptor trafficking. It is unclear whether the regulation of Rho B by hypoxia is dependent on the cellular context or whether hypoxia regulates the expression of Rho B in macrophages.METHODS:For the hypoxic exposure experiment, the cells were incubated in an anaerobic system(1%O 2, 5% CO 2, 94% N 2). Protein and m RNA expression were detected by Western blot and q RT-PCR. Cytokines were detected by ELISA assay. RAW264.7 cells were transiently transfected by lipofectamin 2000 and primary macrophages were transfected by adenoviral infection. The NF-κB luciferase activity was measured by the dual luciferase assay system.RESULTS:Here, we investigated the effect of hypoxia on the expression of Rho B and the mechanism and significance of Rho B expression in macrophages. We found that hypoxia significantly upregulated the expression of Rho B in RAW264.7 cells, mouse peritoneal macrophages,and the spleen of rats.Hypoxia-induced expression of Rho B was significantly blocked by a specific inhibitor of hypoxia-inducible factor-1α(HIF-1α), c-Jun N-terminal kinase(JNK),or extracellular- signal regulated protein kinase(ERK), indicating that hypoxia-activated HIF-1α, JNK, and ERK are involved in the upregulation of Rho B by hypoxia. Knockdown of Rho B expression not only significantly suppressed basal production of interleukin-1beta(IL-1β), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α) in normoxia but also more markedly decreased the hypoxia-stimulated production of these cytokines.Furthermore, we showed that Rho B increased nuclear factor-kappa B(NF-κB) activity, and the inhibition of NF-κB transcriptional activity significantly decreased the Rho B-increased m RNA levels of IL-1β, IL-6, and TNF-α. Finally, we demonstrated that Rho B enhanced cell adhesion and inhibited cell migration in normoxia and hypoxia.CONCLUSION:Upregulation of Rho B promoted the production of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, by activating NF-κB signaling, enhancing macrophage adhesion and inhibiting macrophage migration under hypoxic conditions, all of which sustained the inflammation These findings provide a novel mechanism by which Rho B links macrophage function to the hypoxia-induced inflammatory response. |
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