Study On Anoikis Resistance In Breast Cancer Cells Induced By Kallistatin

Detachment from the matrix induces programmed cell death in endothelial and normal epithelial cells,which is called anoikis.Resistance to anoikis is a critical culprit in the development and progression of tumor.Kallistatin(KST),a member of the serine protease inhibitor family,participates in the regulation of multiple signal transduction pathways through its two functional domains,and plays multiple roles in regulating blood pressure,angiogenesis,oxidative stress,inflammatory response,and programmed cell death.Objectives: To investigate the effects and mechanism of anoikis resistance in human breast cancer cells induced by kallistatin.Methods:(1)Expression of KST and the relationship between the expression level and prognosis in breast cancer were analyzed in TCGA database.(2)MDA-MB-231 and BT549 cells were infected with lentivirus expressing sh KST or sh Control labelled by GFP to construct stable cell lines.(3)Trypan blue exclusion experiment,cluster assay,Annexin-V/633/ PI double staining and Western blot were used to detect apoptosis in stable cell lines under suspension culture condition.(4)Stimulated by exogenous human recombinant KST protein,stable cell lines were detected by cluster assay,Annexin-V,633/PI staining and western blot.(5)The effects of KST on TNF-α-induced anoikis and the potential mechanism were preliminary explored.(6)Transwell migration and invasion assay were used to explore KST effect on cell migration and invasion.Results:(1)TCGA database shows that KST is highly expressed in breast cancer tissues compared to health group,and expression level was associated with poor prognosis.(2)KST knockdown stable cell lines were successfully constructed.(3)Cell cluster was reduced,apoptosis rate was increased and cleaved caspase3 and Cleaved PARP protein expression were increased in KST knockdown stable cell lines compared to the control groups.(4)Stimulated with exogenous human KST protein,stable cell lines’ cluster ability was increased,apoptosis rate was decreased and the expression of cleaved caspase3 and cleaved PARP protein expression was decreased.(5)The apoptosis rate of suspended cultured breast cancer cells increased dose-dependnent of TNF-α,and the phosphorylation level of AKT decreased in a time-dependent manner followed by TNF-α treatment.Cell cluster reduced,apoptosis rate increased and cleaved caspase 3 and Cleaved PARP protein expression increased in KST knockdown stable cell lines compared to the control groups,while exogenous addition of purified KST protein inhibited this effect,and the phosphorylation level of AKT in KST knockdown cells increased.(6)Down-regulation of KST inhibits cell migration and invasion in breast cancer.Conclusion:(1)KST expression was increased in breast cancer compared to the normal group and was associated with poor prognosis.(2)KST promotes anoikis resistance in breast cancer cells.(3)KST may inhibit TNF-α-induced anoikis by promoting AKT phosphorylation in breast cancer.(4)KST may promote cell migration in breast cancer.