Effects Of Pleiotropin （PTN） On The Fak Inhibitor Y15 In Breast Cancer Cells

1,2,4,5-Phenytetramine tetrahydrochloride(C₆H₁₄C_l4N₄)is Y15,a FAK focal adhesion spot kinase inhibitor.FAK is a protein with a molecular weight of 125k Da,localized atcell adhesion spots,activated by integrin clusters and tyrosine phosphorylation.Tyrosine 397,an autophosphorylation site of FAK,is a key component of downstream signaling,providing a high-affinity binding site for the SH2 domain of the Src family kinases.Y15 is an inhibitor that specifically blocks the phosphorylation of Y397-FAK and the total phosphorylation of FAK.Pleiotrophin（PTN）,formerly known as heparin-binding growth-related molecules,is a member of the highly conserved human gene family.PTN exhibits various biological activities as secreted growth factors expressed at high levels in patient sera of several tumors.It indicates that PTN may be a new serum biomarker that can be used to determine tumor burden and to monitor the treatment status ofcancer patients.PTN is both a tumor biomarker and a target for the development of new anticancer drugs.This topic mainly induced the expression of PTN protein from prokaryotic strains with PTN gene,optimized the expression conditions,purified the target protein by nickel-cobalt affinity chromatanalysis using different concentrations of imidazole solution,and used SDS-PAGE electrophoresis and Western blot identification to obtain high purity PTN protein.The protein can promote cell growth and cell migration,and has biological activity.In breast cancer cells MCF-7 and MDA-MB-231 acting with the concentration-gradient kinase inhibitor Y15,The resulting purified PTN protein was added,MTT experiments demonstrated that PTN stimulated greater IC50 values in MCF-7,MDA-MB-231 cells treated with Y15 than in the absent PTN proteome.It showed that PTN protein inhibited the apoptotic effect of Y15 on the cells;MCF-7,MDA-MB-231 cells treated with the kinase inhibitor Y15were stimulated using various concentrations of PTN protein,Apoptosis was measured by flow cytometry.The results showed that PTN protein inhibited apoptosis in a concentration-dependent manner.The effect of PTN protein on Y15 in breast cancer cells was further explored by using the sh PTN interference vector with MCF-7 and MDA-MB-231 for silencing of the PTN gene.Cells were examined for their sensitivity to the kinase inhibitor Y15and for their apoptosis.After 24h after transfection of sh NC,sh PTN1 and sh PTN2,the interference efficiency of sh PTN1 and sh PTN2 at the m RNA and protein levels showed the best interference efficiency of sh PTN2.Comparing the PTN-downregulated breast cancer cells MCF-7 and MDA-MB-231 as compared with normal breast cancer cells,the kinase inhibitor Y15showed a significant reduction in IC50 for its action,that is,to improve the sensitivity to Y15.It was also demonstrated in the flow experiments,the group of MCF-7,MDA-MB-231 cells transfected with the sh NC plasmid,as compared to the Control group,there was no significant changes in apoptosis after the addition of Y15,However,in MCF-7,MDA-MB-231 cells transfected with the sh PTN2 plasmid,Y15induced a significant increase in the apoptosis rate in this group compared with the previous two groups.It was shown that PTN silencing promoted the apoptotic effect of Y15 on breast cancer cells.The results of Western blot experiments showed that Bcl-2,Bax,Cleaved PARP,Cleaved caspase-3,and caspase-3 protein expression was not significantly different after stimulation of MCF-7,MDA-MB-231 cells with PTN protein compared with the control group.In the PTN-silenced MCF-7,MDA-MB-231cells,the expression of Bax,Cleaved PARP and Cleaved caspase-3 proteins increased by Y15 compared with the control group.The Bcl-2 protein expression was significantly reduced compared with the control group,and there were no significant changes in the caspase-3 protein expression levels.Western blot experiments showed that the expression of PI3K（P85 subunit）and p-AKT（S473）phosphorylated protein was significantly higher in MCF-7 and MDA-MB-231 cells were stimulated with PTN protein,and the expression of PI3K（P85 subunit）and p-AKT（S473）phosphorylated protein in MCF-7 and MDA-MB-231 cells silent with PTN was significantly lower after adding Y15.In conclusion,PTN affects the sensitivity of the kinase inhibitor Y15,and intracellular PTN silencing can promote the apoptosis of Y15 on breast cancer cells,and it is speculated that PTN silencing and Y15 may inhibit the survival of breast cancer cells through the AKT/PI3K pathway,which laid the foundation for subsequent in-depth research and clinical treatment of breast cancer.