The Effect Of Doxycycline On Expression Of ERK And Akt Pathway Related Proteins In Myeloma Cell Line

ObjectiveIt has been reported that doxycycline(DOX)had anti-tumor effects on varieties of malignant tumors such as diffuse large B-cell lymphoma,lung cancer,breast cancer,and ovarian cancer.However,it has never been reported that DOX has an anti-myeloma(MM)cell effect.We found in previous experiments that DOX had in vitro anti-myeloma activity,besides,we showed that it downregulates the p-ERK and upregulates the p-Akt a.The mechanisms underlie those phenomenon is still not elucidated.This study try to further investigate the possible mechanisms of ERK and Akt alteration by DOX.Methods1、 The CCK8 method was employed to evaluated the proliferation inhibition effects of DOX on H929 cell line.2、Western Blot method was used to measure the expression of p-Akt,p-ERK1/2,p-MEK protein in the presence or absence of DOX treatment of H929 cells at different time points.3、 The CCK8 method was employed to evaluated the proliferation inhibition effects of U0126 and FR180204 on H929 cell line.Then,the cell proliferation inhibition rate was calculated and suitable inhibitor concentration was screened out.4、The Western Blot method was used to measure the protein expression of p-Akt and p-ERK1/2 in the presence or absence of U0126 treatment of H929 cells at different time points.5、The Western Blot method was used to measure the protein expression of Akt downstream proteins p-GSK-3β and p-BAD in the presence or absence of U0126 treatment of H929 cells at different time points.Results1、Compared with the untreated control group,the proliferation of H929 cells in the DOX-treated group was inhibited.And with the increase of DOX concentration,the proliferation inhibition was showed to be even pronounced.Meanwhile,at a given DOX concentration,the proliferation inhibition become even significant with prolonged time.2、After treatment of H929 cells for 2~3 days by 5mg/L DOX and for 1~3 days by 10mg/L DOX treatment,it was showed that the level of p-ERK1/2 expression was down-regulated,the protein expression of p-Akt(thr308 and ser473)was up-regulated conversely,and the protein expression of p-MEK had no significant changes.3、Compared with the blank control group,after respectively treated by U0126(0,0.625,1.25,2.5,5,10,20,40μmol/L)and FR180204(0,2.5,5,7.5,10,15,20μmol/L)for 1~3 days,the proliferation of H929 cells was inhibited by either agents.As the concentration of either U0126 or FR180204 increased,the inhibition rate are even significant.4 、 After H929 cells has been treated with 10μmol/L U0126 for 1~3 days,it showed that the protein expression of p-ERK1/2 was down-regulated,but the protein expression of p-Akt(thr308)did not change significantly.5、The protein expression of p-GSK-3β and p-BAD was up-regulated in H929 cells treated with 10mg/L U0126 for 1~2 days.Conclusions1 、 DOX was showed to inhibit the proliferation of H929 cells in a time-dependent and dose-dependent fashion.2、DOX may inhibit the proliferation of H929 cells at least partially by inhibiting ERK.3、The up-regulation of p-AKT in H929 cells by DOX is not through the ERK pathway.