| Objective:Doxycycline(DOX)is a commonly used antibiotic.In recent years,the anti-tumor effect of doxycycline has attracted more attention and exploration,but there are no other reports on its effect on myeloma.Our group found in previous experiments that doxycycline can inhibit the proliferation of myeloma cells and is accompanied by down-regulation of p-Erk1/2protein.The reason and mechanism are still unclear.This experiment studies the effect of doxycycline on the apoptosis of myeloma cell lines under different conditions,and explores the possible mechanism of the Erk pathway that doxycycline may play in the process of inducing the apoptosis of myeloma cell lines(H929).Method:1.Western Blot method was used to detect the expression of apoptosis-related proteins in H929 cells treated with doxycycline.Designed to treat H929 cells with 10mg/l DOX for 1,2,and 3 days,and treat H929 cells with different concentrations of DOX(5mg/l,10mg/l,15mg/l)for 3 days;the drug-free treatment group was used as the control group.2.Western Blot method was used to detect the effect of different concentrations of DOX on the expression of Erk protein and its upstream MEK protein in H929 cells.The concentrations of DOX were 5mg/l,10mg/l,15mg/l,and each experimental group was treated for 3 days.3.Western Blot method was used to detect the effects of different concentrations of U0126(MEK inhibitor)on the expression of MEK and Erk proteins after treating H929 cells.The concentration of U0126 is 2u M,4 u M,6 u M,and the treatment time is 3 days.4.The CCK8 method was used to detect the Ras agonist(ML-098)combined with Dox to treat H929 cells to detect its effect on cell proliferation.The control group was treated with no drug.Detect the absorbance value of each group with a microplate reader,and calculate the cell proliferation inhibition rate of each experimental group.The experimental design is divided into control group,DOX group,DOX combined with ML-098 group;treatment for 1,2,and 3 days respectively.5.The Annexin-V/PI double staining method was used to detect the apoptosis of H929 cells after ML-098 combined with Dox treatment.The experiment is divided into 3 groups: control group,DOX single-drug group,DOX combined with ML-098 group.6.H929 cells were treated with 0.5u M ML-098 combined with 10mg/L Dox for 2days,and Western Blot method was used to detect the expression of p-Erk1/2,p-MEK,activated caspase-3,activated caspase-9 and other proteins.The experiment was designed into 3 groups: control group without any drug treatment,group with 10mg/l DOX culture for 2d,0.5u M ML-098 combined with 10mg/l DOX culture for 2d group.7.RT-PCR was used to detect the expression levels of c-Fos and c-Jun genes regulated by the Erk pathway.Experimental design: DOX concentration was 10 mg/L,treated for 1,2,and 3 days respectively.8.After DOX treatment of H929 cells,Western Blot method was used to detect the expression of c-Jun protein.Results:1.Different concentrations of DOX treated H929 cells for 3 days,and 10 mg/l DOX treated H929 cells for different days,Western Blot results showed that the expression of activated caspase-3 and caspase-9 proteins in each experimental group increased compared with the control group(P<0.05).2.Compared with the control group,the expression of p-MEK protein decreased after doxycycline treatment of H929 cells(P<0.05).3.After MEK inhibitor(U0126)treated H929 cells,the expression of p-MEK protein decreased(P<0.05),and the expression of activated caspase-3 and caspase-9 proteins increased(P<0.05)in each experimental group.4.After DOX combined with ML-098(Ras agonist)treated H929 cells for 2 days,the apoptosis results showed that the apoptosis in the two-drug combination group was lower than that in the DOX group;and Western Blot results showed that the two-drug combination group p-The expression of MEK and p-Erk protein was higher than that of DOX(P<0.05),and the protein expression was lower than the drug-free control group(P<0.05);the expression of activated caspase-3 and 9 proteins was lower than that of the DOX group(P<0.05).0.05),and higher than the control group(P<0.05).5.After DOX treatment of H929 cells,Real-time-PCR results showed that: c-Jun gene m RNA expression level was up-regulated(P<0.05),c-Fos gene m RNA expression level was down-regulated(P<0.05);and DOX treatment H929 cells c-The expression of Jun protein increased(P<0.05,compared with the drug-free treatment group).Conclusions:1.DOX induces apoptosis of the H929 cell line,and the Erk pathway plays an important role in the process.2.The apoptosis of H929 cells induced by DOX was mediated by the intrinsic caspase pathway.3.DOX causes the up-regulation of c-Jun protein in H929 cells and promotes apoptosis.4.In DOX-induced apoptosis of H929 cell line,p-Erk may have a direct or indirect effect on c-Jun. |
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