| Objective:Doxycycline(DOX)is a tetracycline antibiotic.In addition to anti-infective effects,it has also been found to have certain anti-tumor effects in recent years.Our previous study found that DOX could inhibit the proliferation and induce apoptosis of human myeloma cell lines.In addition,the expression level of c-Jun protein was increased during DOX treatment,but the mechanism and effects of c-Jun up-regulation induced by DOX remains unexplored.Besides,some studies have found that DOX also demonstrated glycolysis modulation effects in some types of tumor,but its role on glycolysis in myeloma has never been reported.For the above reasons,in the present study,myeloma cell line H929 was used to explore the effect of DOX on JNK pathway and glycolysis,as well as the effect of JNK pathway on cell proliferation,apoptosis and glycolysis.Method:1.The effect of DOX treatment on the expression of p-JNK protein in H929 cells was measured by Western Blot.Both 5mg/l and 10mg/l of DOX were employed as concentration for the in vitro experiment,and the treatment duration was 2 days;No drug treatment group was the negative control group.2.The effect of JNK inhibitor AEG3482 on the proliferation of H929 cells was measured by CCK8 method.A blank control group was set up in the experiment and the negative control group without any drug treatment was selected.OD value(absorbance value)of each group was detected,cell proliferation inhibition rate was calculated,and cell proliferation inhibition curve was drawn.The concentrations of AEG3482 were 5μmol/L,10μmol/L,15μmol/L and 20μmol/L,respectively.They were treated for 1,2 and 3 days respectively.3.The effects of 20μmol/L AEG3482 combined with 10mg/L DOX on the proliferation of H929 cells were determined by CCK8 method.A blank control group was set up,and a negative control group without any drug treatment was selected.The absorbance of each group was detected and the inhibition rate of cell proliferation was calculated.The experimental design was divided into four groups: blank control group,no drug treatment group,single DOX group,DOX combined with AEG3482 treatment group;They were treated for 1,2 and 3 days respectively.4.The apoptosis of H929 cells was measured by FCM while treated with AEG3482.The concentrations of AEG3482 were 10 u M,15 u M and 20 u M and the treatment duration was 2 days;No drug treatment group was the negative control group.5.The apoptosis of H929 cells was measured by FCM while treated with 20 u M AEG3482 combined with 10mg/L DOX for 2 days.The experiment was divided into three groups: control group,single DOX group and DOX combined with AEG3482 treatment group.6.Western Blot was used to detect the effects of AEG3482 on the levels of p-JNK and cleaved caspase-3 protein in H929 cells.The concentrations of AEG3482 were 10 and 20 u M and the treatment duration was 2 days;No drug treatment group was the negative control group.7.H929 cells were treated with DOX combined with AEG3482 for 2 days,and the levels of p-JNK and cleaved caspase-3 protein were detected by Western Blot.The experimental design was divided into three arms: no drug treatment arm,single DOX treatment arm,and 20 u M AEG3482 co-treatment group with 10mg/L DOX.8.The concentrations of lactic acid produced by H929 cells after treated with DOX were detected by lactic acid detection kit.DOX concentration was 5 and 10mg/l,respectively,and the treatment time was 2 days.A blank control group was set up,and the group without any drug treatment was the negative control group.9.Western Blot was used to detect the effect of DOX on the levels of HK2,PKM2,PFKP(key glycolysis enzymes)protein in H929 cells.DOX concentration was 5mg/l and 10mg/l,respectively,and the treatment time was 2 days.No drug treatment group was the negative control group.10.Lactic acid kit was used to detect the concentrations of lactic acid produced by H929 cells after being treated with AEG3482 for 2 days.The concentrations of AEG3482 were 10、15 and 20μmol/L.A blank control group was set up,and the group without any drug treatment was the negative control group.11.The effect of different concentrations of AEG3482 on HK2 m RNA expression in H929 cells was detected by Real-Time RT-PCR.The concentrations of AEG3482 were 10、15 and 20μmol/L and the treatment duration was 2 days.No treatment arm was the negative control group.12.Western Blot was used to measure the effect of AEG3482 on the level of HK2 protein in H929 cells.The concentrations of AEG3482 were 10 and 20 u M and the treatment duration was 2 days.No drug treatment group was the negative control group.13.H929 cells were treated with DOX combined with AEG3482 for 2 days,and the changes of lactic acid content were detected with lactic acid kit.The experimental design was divided into four arms: blank control arm,no drug treatment arm,single DOX treatment arm of 10mg/L,10mg/L DOX combined with 20 u M AEG3482co-treatment arm.14.The effect of DOX combined with AEG3482 on the expression of HK2 protein in H929 cells for 2 days was detected by Western Blot.The experimental design was divided into three arms: no drug treatment arm,single DOX treatment arm of 10mg/L,and 20 u M AEG3482 co-treatment group with 10mg/L DOX.Results:1.After treating H929 cells with DOX at different concentrations for 2 days,the expression of p-JNK protein was up-regulated(P<0.05,compared with control group).2.Compared with the control group,AEG3482 at the concentration of 10-20 u M had obvious inhibitory effect on H929 cell proliferation after 1,2 or 3 days’ treatment;After AEG3482 combined DOX treatment with H929 cells for 1,2 or 3days,the results of CCK8,showed that the cell proliferation inhibition rate of 20 u M AEG3482 co-treatment group with 10mg/L DOX was increased compared with that of single DOX treatment group(P<0.05).3.After 2 days of treatment with different concentrations of AEG3482,flow cytometry showed that compared with the control group,the apoptosis ratio of H929 cells in each experimental group was increased(P<0.05).Western Blot results showed that compared with the control group,the expression of p-JNK protein was decreased(P<0.05),while the expression of cleaved caspase-3 protein was increased(P<0.05).4.H929 cells were treated with DOX combined with AEG3482 for 2 days,and flow cytometry results showed that in the combined treatment arm,the apoptosis ratio of H929 cells was increased(P<0.05),when compared with single DOX treatment arm;Western Blot results showed that compared with DOX treatment group,p-JNK protein expression was decreased(P<0.05),and caspase-3 protein expression was increased(P<0.05).5.After treating H929 cells with DOX at different concentrations for 2 days,lactic acid production and HK2 protein expression were significantly decreased when compared with the drug-free treatment group(P<0.05),while the protein levels of PFKP and PKM2 were not significantly altered.6.After 2 days of treatment with different concentrations of AEG3482,the lactic acid production,HK2 m RNA and protein expression levels were decreased(P<0.05,compared with no drug treatment group).7.H929 cell line was treated with DOX combined with AEG3482 for 2 days,the lactic acid production and HK2 protein expression were significantly decreased,when compared with DOX treatment arm(P<0.05).Conclusions:1.DOX up-regulated JNK signaling pathway and inhibited glycolysis in myeloma H929 cells.HK2 may play a key role in the inhibition of glycolysis in H929 by DOX.2.Inhibition of JNK signaling pathway could inhibit the proliferation of H929 cell line and induce its apoptosis.At the same time,glycolysis was also inhibited.3.The Inhibition of JNK pathway together with DOX treatment was showed to enhance its anti-proliferation,pro-apoptosis and anti-glycolysis effects of DOX in H929.4.Our results showed that JNK pathway up-regulation had a negativle role in its anti-myeloma effects during DOX treatment,and the inhibition of JNK pathway together with DOX treatment could enhance its anti-myeloma effects. |
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