The Biomechanical Mechanisms Of Shear Stress Mediated-upregulation Of Cav-1 Expression And Regulation Of Anoikis Resistance Of Breast Cancer Cells

The Breast cancer is the leading cause of cancer mortality worldwide,and most death is associated with tumor metastasis.To metastasize,a malignant cell must detach from the extracellular matrix,invade the nearby circulatory or lymph system,and establish itself in a new site.Once in the bloodstream,most of the cells die by anoikis.However,some cancer cells develop resistance to anoikis and consequently survive to establish new metastases.Caveolin-1(Cav-1)is an essential protein constituent of the plasma membrane caveolae.As a mechanical receptor,Cav-1 can feel shear stress from blood and tissue fluid,regulate various signal pathways,and affect proliferation,migration and apoptosis of cancer cells.However,the effects of Cav-1 on anoikis resistance remain poorly understood.Previous studies suggested that shear stress induced the up-regulation of Cav-1 expression.However,the specific mechanism by which shear stress increases the expression level of Cav-1 are unknown.Therefore,this paper intends to explore how the shear stress increases the expression level of Cav-1,and how does the increased expression level of Cav-1 affects anoikis resistance ability of tumor cells and the relevant molecular mechanism.In this study,we found that the expression level of Cav-1 can be increased after the cells were treated with shear stress(2 dyn/cm~2)and suspended culture for 48 h through the result of Western Blot.Then,by using immunofluorescence staining,flow cytometry and Western blot,we observed the changes of growth rate,apoptosis rate and the expression level of apoptotic proteins in shCtrl and shCav-1 cells.The experimental results showed that the apoptotic rate of cells decreased and the expression level of pro-apoptotic proteins was decreased and the expression level of the anti-apoptotic protein was increased after shear stress treatment in shCtrl cells.In contrast,the apoptotic rate and the expression of the associated apoptotic proteins with or without loading shear force Neither was significantly changed in shCav-1 cells,moreover,the proliferation rate was significantly lower than that the group of shCtrl cells,and the apoptosis rate was significantly higher than that the group of shCtrl cells.It suggests that shear stress is responsible for enhancing cancer cells’resistance anoikis by promoting Cav-1 expression.Subsequently immunofluorescence experiments proved that low shear stress induces nitric oxide or reactive oxygen species generation.The scavenging of nitric oxide or reactive oxygen species induced by shear stress,then the apoptotic rate of cells increased,causes that the expression level of Cav-1 increased,which suggests that nitric oxide or reactive oxygen species induced by shear stress can promote the expression of Cav-1,and resulted in the ability to resist anoikis.Finally,immunoprecipitation experiments proved that nitric oxide or reactive oxygen species induced by shear stress inhibited the proteasomal degradation pathway of Cav-1,thereby increasing the expression level of Cav-1 in cells.Conclusion:Shear stress inhibits Cav-1 proteasomal ubiquitylation pathway by increasing the level of nitric oxide or reactive oxygen species,which increases the expression level of Cav-1 and promotes anoikis resistance of human breast cancer cells.