Study Of Notch Signaling Inhibitor DAPT On Proliferation、 Apoptosis And Chemotherapy Sensitivity In RM-1Cells Of Prostate Cancer Bone Metastases

objective:1、 Using mice prostate cancer RM-1cells for research object, to investigatethe effect of DAPT(γ-secretase inhibitor) on the proliferation、apotosis andchemotherapy sensitivity of RM-1cells by inhibiting Notch signaling pathwaysin vitro.2、 Establish a bone metastasis model of prostate cancer by intratibia injection ofRM-1cells in mice,studying the effect of DAPT on metastatic bone tumorgrowth and the chemotherapy sensitivity in vivo.Methods:1、 RM-1cells were randomly divided into two groups: the experiment group inwhich specimens were treated with DAPT at the concentration of0.25、0.5、1、2、5μmol/L, and the control group in which specimens were treated with medium;Cell proliferation was determined using MTT method;The cell cycle wereanalyzed by flow cytometry（FCM）with PI staining method;The cell apoptosiswas detected by Tunel method;RT-PCR、Western blot was used to confirm theexpression of Notch1、Jagged1mRNA and protein.RM-1cells was pretreatedwith DAPT（1μ mol/L）for24h and without DAPT, the changes of IC50of ADMwas detected by MTT method.2、 Tibia intramedullary injection3weeks later，Using X-ray, three-dimensional CTreconstruction, HE dyeing methods assessment model；Model was randomlydivided into four groups：control group、DAPT group、ADM group、ADM+DAPT group， with Corresponding treatment four weeks later， tumororganizations was stripped carefully to weighing wet weight.Results:1、 compared with the control group,0.25umol/L DAPT group has no significant effect on proliferation、 the cell cycle distribution、 the apoptosis and theexpression of Notch1、Jagged1（P>0.05）.0.5,1,2,5μ mol/L DAPT group cansignificantly suppress cell proliferation、induce cell G0/G1cycle arrest、promote apoptosis and down-regulate the expression of Notch1and Jagged1in adose dependent manner(P <0.05). The IC50of ADM was (6.25±0.38)μg/ml，while pretreated with1μ mol/L DAPT for24h，the IC50of ADM was(3.25±0.52)μg/ml.2、 Tibia intramedullary injection3weeks later，X-ray, three-dimensional CTreconstruction shows bone destruction.After dissection,some "fish-like” tissuegrew from marrow cavity to the muscle in invasive growth manner, and theboundary is vague.The "fish-like” tissue was identified to tumor by HE. Afterintraperitoneal injection four weeks, the tumor wet weight of control group、DAPT group、ADM group, ADM+DAPT groups were（3.45±0.22）g、（3.21±0.37）g、（2.87±0.28）g、（2.25±0.41）g，there were significant differencesbetween groups.Compared with control group,DAPT group had obviousdifferences (P <0.01); Compared with ADM group,ADM+DAPT group hadobvious differences also (P <0.01).Conclusions:1、 When the concentration of DAPT is0.5μ mol/L to5μ mol/L,DAPT cansignificantly suppress cell proliferation、induce cell G0/G1cycle arrest andpromote apoptosis in dose dependent manner in vitro, the mechanisms may beassociated with the down-regulation of Notchl、Jagged1mRNA and proteinexpression.2、 Bone metastasis model of prostate cancer is established successfully by intratibiainjection of RM-1cells in mice.3、 DAPT has the role of suppress tumor growth in vivo.4、 DAPT has a role of raising chemotherapy sensitivity of ADM in vivo and in vitro.