Tetracyclines Doxycycline Exhibit Antimyeloma Effect Involving Stat,Erk And Akt Pathways

Objective: Doxycycline(DOX)is a commonly used tetracycline antibiotic,and its anti-myeloma effect has not yet been reported.This study was to investigate antimyeloma effect of Doxycycline(DOX)and its possible mechanism.Methods:1,MTT assay was employed to assay DOX’s anti-myeloma effect on human myeloma cell lines H929 and U266.A total of 6 concentration gradient were 1.25 mg/L,2.5 mg/L,5 mg/L,10 mg/L,20 mg/L,40 mg/L and a total of 3 time gradient were 24,48 and72 hours,using MM without any treatment as negative control,and a blank control group(no adding).The OD value was measured and the cell proliferation inhibition rate was calculated.2,Western blot was employed for the detection of PARP,phospho-Stat1(p-Stat1),phospho-Akt(p-Akt)and phospho-p44/42MAPK(p-Erk1/2)of MM treated by DOX.There were four groups in this study: 1)group A: MM cell lines without any treatment as control;2)group B: MM with 2.5mg/L DOX effect after 3 days;3)group C: MM with 5mg/L DOX effect after 3 days;4)group D: MM with 10mg/L DOX effect after 3 days.3,MTT assay was used to determine the effect of the combination of DOX and Akt inhibitor perifosine on the proliferation of human myeloma cell lines U266 cells.Fristly,U266 cells were treated by Perfosine alone for 4 hours,then all the perfosine was removed from the culture and U266 was further cultured for additional 3 days as a control.Secondly,U266 cells were first treated by perifosine for 4 hours,and then U266 cells harvested and washed away any perifosine contamination,and transferred to 5mg/L DOX-containing medium for 3 days.The OD value was measured and the cell proliferation inhibition rate was calculated.4,Western blot was employed for the detection of phospho-Akt(p-Akt)of U266 cell lines treated by different ways of Perifosine treatment.1,negative control group.2,U266 cell line was treated by Perfosine for 4 hours and then the expression of p-Akt was assayed by western blot.3,U266 cell line was treated by Perifosine 4 hours,and then cultured for 3 days without perifosine.4,U266 cell line was continuously treated by 5μM Perifosine for 3 days.5,Western blot was employed for the detection of phospho-Akt(p-Akt)of U266 cell lines treated by DOX alone or DOX combined with Perifosine.1,no drug treatment group was negative control group;2,5mg/L DOX was employed to treated U266 cell line for 3 days alone;3,U266 cell line was treated bywith Perifosine for 4 hours,and then treated with DOX alone for 3 days.The expression of p-Akt protein in U266 cells was assayeddetected to to investigate whether Perifosine can inhibit the up-regulation of p-Akt expression by DOX.6,The total RNA of the four groups of MM cells after the DOX treatment were collected and extracted.The mRNA expression of c-Fos and c–Jun in U266 cells were measured by Real-time PCR,and further statistically analyzed.Results:1,The proliferation of both myeloma cell lines was showed to be inhibited by the DOX treatment in a time-and dose-dependent manner(P<0.05).2,DOX suppressed the expression of p-Erk1/2 in both MM cell lines in dose dependent manner(P<0.05).DOX was only showed to up-regulation of p-Stat1 in H929(P<0.05).Compared to the no treatment control group,there was no dramatic difference in the expression level of p-Stat1 in U266 and PARP in both H929 and U266.And the expression of p-Akt was up-regulated in both cell lines,and this effect could be reversed by Akt inhibitor Perifosin(P<0.05).Perifosine inhibited the action of Akt irreversibly,and MTT results also suggested that Perifosine enhanced the toxic effects of DOX on U266 cell lines.3,The mRNA expression levels of c-Fos in U266 cell was down-regulated after DOX treatment(P<0.05).Conclusion: To the best of our knowledge,this study is the first one showing DOX exhibit anti-myeloma effect and Stat,Erk and Akt pathways might be involved in its effects on myeloma cell lines.