The Effect Of Calcitonin Gene-related Peptide And CGRP Receptor Antagonist On Bone Metastasis Microenvironment Of Breast Cancer In Vitro

Objective and Aim(1). The study is aimed to observe the effects of calcitonin gene-related peptide(CGRP)and CGRP receptor antagonist CGRP8-37on the expression of osteoclastic regulatorfactors of osteoblast through osteoblast-like cells cultured alone in vitro.(2）.The study is aimed to observe the effects of calcitonin gene-related peptide(CGRP)and CGRP receptor antagonist CGRP8-37on the expression of osteoclastic regulatorfactors of osteoblast through in vitro coculture model of breast cancer cells andosteoblast-like cells.Method(1).In the first part of the experiment, MG-63cells were planted at a density of3×105cells/well alone with DMEM media containing10%FBS. The medium waschanged4-6hours later to another medium not containing FBS. After12hours mediumwas changed again. Two groups were treated with CGRP10-8M or with CGRP8-3710-8M respectively. The cells without CGRP and CGRP8-37treatment served ascontrol. We tested the mRNA and protein expression of osteoprotegerin(OPG),receptoractivator of NF-κβ ligand(Rankl) and runt-related transcription factor2(Runx2).(2). In the second part of the experiment,MG-63cells were plated with MDA-MB-231at a density of5×105cells/well in6-well plates with DMEM media containing10%FBS.Twogroups were treated with CGRP10-8M or with CGRP8-3710-8M respectively. TheMG63cultured alone and coculture model without CGRP and CGRP8-37treatment areserved as control. We tested the mRNA and protein expression ofosteoprotegerin(OPG),receptor activator of NF-κβ ligand(Rankl) and runt-related transcription factor2(Runx2).Result(1). When MG63was cultured only，OPG expression was significantly increased byCGRP，while the expression of Rankl and Runx2were down-regulated by CGRP(p﹤0.05).CGRP8-37significantly inhibited the CGRP-increased OPG expression,except RANKLand Runx2(p﹤0.05)．(2).When cocultured with breast cancer cells,the expression of Rankl and Runx2wereup-regulated at both mRNA and protein level,while the expression of OPG wasdown-regulated(p﹤0.05).These up-regulations of Rankl and Runx2and down-regulationof OPG could be reversed by CGRP treatment(p﹤0.05).CGRP8-37can inhibite thiseffect(p﹤0.05)．Conclusion（1）CGRPcan up-regulate the expression of OPG and down-regulate the expressionof RANKL and Runx2.（2）MDA-MB-231promote osteoclast via regulating the osteoblast activity bychanging the expression of OPG-Rankl,and meanwhile they inhibit the activity ofosteoblast cells through regulation of Runx2expression.（3）As a competitive inhibitor of CGRP,CGRP8-37can be used extensively to probeand elucidate the actions of CGRP.（4）CGRP may become a potential therapeutic agent for treatment of breast cancerwith bone metastasis through OPG/Rankl.