Effects Of Paclitaxel Alone Or In Combination With Sorafenib On The FLT3-ITD Mutation AML Cell Lines

Background:Acute myeloid leukemia（AML）is a hematological malignancy that is most common in adults.Studies have shown that most AML patients often have a variety of gene mutations closely related to the occurrence,development and prognosis of leukemia,among which the incidence of AML patients with FLT3-ITD mutations that is an important therapeutic target,is as high as 30%,and often has a poor prognosis.It was therefore reasonable that more and more studies have focused on the treatment of AML patients with the development and use of small molecule inhibitors targeting FLT3.However,the dose-toxicity of FLT3 inhibitors limits their responses,and most importantly,the secondary mutations of FLT3 often result in acquired resistance which easy to make the patients relapse after remission.This greatly limits the application of FLT3 inhibitors.Thus,the above of problems highlight the important to find new drugs or treatment strategies for the AML patients.Paclitaxel,with unique anti-cancer action,is considered to be one of the most successful natural anti-cancer drugs at present.Previous studies have shown that paclitaxel can inhibit the proliferation of leukemia cell lines and induce cell apoptosis,such as HL-60,K562 and so on,illustrating a good anti-leukemia effect of paclitaxel.However,whether paclitaxel also has inhibitory effect in the AML cell lines MOLM-13 and MV4-11with FLT3-ITD mutation is unknown.Therefore,clarifying the inhibition of proliferation and apoptosis inducing of paclitaxel in FLT3-ITD mutation AML cell lines MOLM-13、MV4-11,and elaborating the mechanism of action were the main purposes of this study.Moreover,we will also preliminarily investigate the inhibitory effect and the mRNA expression of pro-apoptotic genes such as caspase-3,bax and bid of paclitaxel combined with sorafenib on MOLM-13 and MV4-11 cells.Objective:To investigated the effects of paclitaxel alone on the proliferation and apoptosis of FLT3-ITD mutation AML cell lines MOLM-13 and MV4-11and the relationship between the inhibitory effect of paclitaxel and PI3K/Akt/mTOR signaling pathway.To observe the effects of paclitaxel combined with sorafenib in the proliferation、apoptosis and the mRNA expression of pro-apoptotic genes such as caspase-3,bax and bid of MV4-11and MOLM-13 cells.Methods:Using MOLM-13 and MV4-11 cell lines as experimental subjects,THP-1 and K562 without FLT3 mutation as negative control lines,and sorafenib as positive control,and then treating the above four cell lines with different concentrations of paclitaxel for different times,observing and comparing the effects of paclitaxel on proliferation and apoptosis of MOLM-13 and MV4-11cell lines.The expression levels of FLT3 gene and key genes and proteins in PI3K/AKT/mTOR signaling pathway were detected by PCR and Western blot after treatment with paclitaxel for 48 hours.MV4-11 and MOLM-13 cell lines were treated with paclitaxel combined with PI3K/mTOR pathway inhibitor PKI-587 for 48 hours,and then detecting the cell activity and apoptosis rate and calculating the synergistic index q value to judge the combined effect of the two drugs,in order to investigate the mechanism of paclitaxel inhibiting the growth of MV4-11 and MOLM-13 cells.MV4-11 and MOLM-13 cell lines were treated with paclitaxel and sorafenib for 48 hours to detect the cell activity and apoptosis rate and calculate the synergistic index q value to judge the combined effect of the two drugs,for further verifying the relationship between the inhibition of paclitaxel on MV4-11 and MOLM-13 cell lines and PI3K/Akt/mTOR signaling pathway.Meanwhile,PCR assay was used to detect the mRNA expression of pro-apoptotic genes such as caspase-3,bax and bid.Results:1.Paclitaxel has obvious inhibitory effects on the proliferation and induction of apoptosis of MOLM-13 and MV4-11 cell lines,and with the extension of action time and the increase of drug concentration,the inhibitory effect of paclitaxel on the proliferation of cells was gradually enhanced,in a typical time and concentration dependence.2.Compared with THP-1 and K562 cell lines,paclitaxel had lower IC₅₀value and higher apoptosis rate on MV4-11 and MOLM-13 cell lines（P<0.05）;Compared with sorafenib,the IC₅₀ value of paclitaxel on MV4-11 and MOLM-13 cell lines was also lower（P<0.05）.3.After treated with paclitaxel for 48 hours,the mRNA expression levels of FLT3,PI3K,Akt,mTOR and S6K genes and the protein expression levels of Akt,p-Akt,S6K and p-S6K in cells were decreased（P<0.05）.The use of PKI-587inhibitor could increase the proliferation inhibition rate and apoptosis rate of paclitaxel（P<0.05）,and the combination of the two drugs had a synergistic effect.4.The combination of paclitaxel and sorafenib had obvious inhibitory effect on cells,compared with the single drug group,the proliferation inhibition rate and apoptosis rate of cells were higher（P<0.05）,and the combination of the two had an additive effect.After combined treatment with paclitaxel and sorafenib,the mRNA expression of pro-apoptotic genes such as caspase-3,bax and bid in cells were higher than those in monotherapy group（P<0.05）.Conclusion:1.Paclitaxel can significantly inhibit the proliferation of MV4-11 and MOLM-13 cell lines and induce cell apoptosis,and the possible molecular mechanisms are down-regulation of FLT3 gene expression and inhibition of PI3K/Akt/mTOR signaling pathway activity.2.The combination of paclitaxel and sorafenib can significantly inhibit the proliferation of MV4-11 and MOLM-13 cells,significantly increase the apoptosis rate,and can also up-regulate the mRNA expression of pro-apoptotic genes such as caspase-3,bax and bid,and the combination of them has an additive effect.