The Effect Of Doxycycline On PI3K/Akt Pathway In Myeloma Cells And Its Related Mechanism

ObjectiveDoxycycline(DOX)is one of the most common antibiotics in clinical practice,and its anti-tumor effect has attracted much attention in recent years.However,the effect and the mechanism of DOX on myeloma cells are still unclear.In the previous experiments,we found that cell proliferation was inhibited and the expression of p-Akt was up-regulated with the treatment of DOX.The mechanism was currently not clear.In this study,we firstly detected the levels of p-Akt expression at different time or different concentration in H929 cells during DOX treatment.Secondly,we studied the possible mechanism of the up-regulation of Akt and the downstream effects of Akt pathway up-regulation.Methods1?CCK8 was employed to detect the cell proliferation of H929 cells treated with different concentration of DOX(0~40mg/L)for 1~3d.Western Blot was used to detect the expression of p-Akt at different time of H929 cells treated with 5mg/L and 10mg/L DOX.2?Western Blot was used to detect the expression of p-PDK1 and PTEN of H929 cells treated with DOX.The concentration of DOX is 5mg/L and the time was 3h?6h?10h?1d?2d?3d?1 day post-treatment?2 day post-treatment.3?CCK8 was employed to detect the cell proliferation of H929 cells treated with different concentration of Wortmannin(0~2000nmol/L)for 1~2d.Then we selected the suitable concentration.Western Blot was used to detect the expression of p-Akt of H929 cells with this concentration.4?CCK8 was used to detect the cell proliferation of H929 cells treated with the combination of DOX and Wortmannin.Western Blot was used to detect the expression of p-Akt of H929 cells treated with the combination of DOX and Wortmannin.There was four groups: no treatment,5mg/L DOX 2d,1000nmol/L Wortmannin 4h,5mg/L DOX 2d+1000nmol/L Wortmannin 4h.5?Western Blot was used to detect the expression of p-mTOR? p-GSK-3? and p-Bad of H929 cells treated with 10mg/L DOX.RT-PCR was used to detect the mRNA levels of mTOR,Bcl-2 and NF-?B of H929 cells treated with DOX(0~10mg/L)for 3d.6?ELISA was used to detect the concentration of EGF and IGF-1 in culture medium of H929 cells treated with 5mg/L DOX.The time was 16h?1d?2d and 3d.7?Western Blot was used to detect the expression of p-Erk1/2 of H929 cell treated with 10mg/L DOX or 1000nmol/L Wortmannin.Results1?The cell proliferation of H929 was inhibited with the treatment of DOX for 1~3d.And the inhibition rate increased with the increasement of drug concentration and treatment time.Different concentrations of DOX could up-regulate the expression of p-Akt in H929 cells.2?The up-regulation of p-PDK1 of H929 cells was detected at 3h and 6h with the treatment of 5mg/L DOX,but no change was observed at other time points.PTEN was unchanged at all time points.3?The cell proliferation of H929 cells was inhibited with the treatment of Wortmannin for 1~2d.1000nmol/L was selected as the concentration of the following experiment.The down-regulation of p-Akt of H929 cells was detected at 3h?4h and 6h.We selected 4h as the time of the following experiment.4?The cell proliferation of H929 cells was inhibited with the treatment of 5mg/L DOX for 2d as well as 1000nmol/L Wortmannin for 4h.Cell proliferation rate was significantly reduced with the treatment of the combination of two drugs,showing a synergistic effect.The up-regulation of p-Akt of H929 cells was detected with the treatment of 5mg/L DOX.And the down-regulation of p-Akt of H929 cells was detected with the treatment of 1000nmol/L Wortmannin.The combination experiment suggested that Wortmannin significantly down-regulated p-Akt activated by DOX.5?With the treatment of 10mg/L DOX,there was no significant change in the expression of p-mTOR,but the expression of p-Bad and p-GSK-3? were up-regulated.The genes mTOR?Bcl-2?NF-?B were all down-regulated with the treatment of different concentration of DOX for 3d.6?We detected the change of EGF and IGF-1 in culture medium with the treatment of 5mg/L DOX.There was no significant change in the expression of EGF.However,the expression of IGF-1 was up-regulated.7?The down-regulation of p-Erk1/2 of H929 cells was detected with the treatment of 10mg/L DOX or 1000nmol/L Wortmannin.Conclusions1?Our results showed that DOX could inhibit the proliferation of H929 cells in a time-and dose-dependent manner(P<0.05).2?The mechanism of p-Akt up-regulation in H929 cells may be related to the cellular stress response to DOX cytotoxic effects.This up-regulation effects could be inhibited by the PI3 K inhibitor Wortmannin,suggesting that the mechanism of the up-regulation of p-Akt is at least partially mediated by the PI3K/Akt pathway.3?The expression of p-Bad and p-GSK-3? were up-regulated of H929 cells with the treatment of DOX,it may be related to the downstream activation effects of p-Akt.The mRNA of mTOR,Bcl-2,NF-?B were down-regulated,it may be related to the inhibition of DOX on H929 cells.4?The expression of IGF-1 in supernatant was increased after DOX treatment of H929 cells,which may be related to the secretion of IGF-1 by H929 cells.5?The expression of p-Erk1/2 was down-regulated of H929 cells with the treatment of DOX,it may result from the cross-activation between the PI3K/Akt and Ras/ERK pathway.