Effects Of Indomethacin On Proliferation And Senescence Of Leukemia Cells

Objective: It is reported that the COX-2 is up-regulated expression in a variety of cancers including leukemia recently. Meanwhile,it is believed that its up-regulated expression is associated with the tumor cell proliferation,adhesion and so on. However, NSAIDs can suppress the activity of cyclooxygenase.So they may also have inhibitory effect on tumors.The study, observing the effects of indomethacin on proliferation and senescence of leukemia cells, was aimed to analyze whether non-steroidal anti-inflammatory drugs have any adjunctively therapeutic effects on leukemia.Methods: 1. Cell culture and group intervention Three kinds of leukemia cell lines(U266, K562 and U937) were selected in this experiment. All of the cell lines were cultured in IMDM medium containing 10% fetal bovine serum(FBS) at 37℃with 5%CO2. The logarithmic phase cells were used for following experiments. After the cell concentration were adjusted to 106/ml per well, each kind of cell line was randomly divided into two groups: with the use of indomethacin(final concentration 30μM) as intervention group and the same volume of DMSO as control group. 2. Cell proliferation inhibition test(Typan Blue Exclusion) Use the logarithmic phase cells and inoculate them to 24 well culture dish with cell concentration 104/ml. Make the final concentration 30μM of indomethacin as intervention group and the same volume of DMSO as control group.Set four parallel holes each group. We collect the cells cultured(0,4,7)days.Centrifuge and dilute them into single-cell suspension.Check it by Typan Bule Exclusion. Check the cells by cell counting chamber within 5 minutes. We define that the unpigmented are living cells and colored by Typan Blue are death cells under the light microscopy. The survival cell(%)=living cells/(living cells + death cells)*100%.3. Determination of the cell cycle Dilute all kinds of cells to 104/ml by medium according to the different groups.Wash them once by PBS(phosphate belanced solution).Then add it with 70% ethanol and put it in refrigerator at 4℃ overnight. Mark cells by the protocol of the cell cycle kit. We use the flow cytometry to check the cell cycle and detect the phase change of the different groups. 4.Apoptosis assay We check the cells cultured for 4 days by Annexin V-FITC/PI kit. All the groups are determined by FCM. 5.Cell senescence Collect all kinds of cells cultured for(0,4,7)days. Wish them with PBS.We check it on the light microscopy according to the protocol of Cell senescence kit. Cell senescence(%)=(the total number of senescent cells/the total cells)*100%. 6.RT-PCR detection(p21,p27 m RNA) Choose the logarithmic phase cells and dilute them to 104/ml by medium.Collect the cells cultured for 4 days.Extract the total RNA by High Pure PCR Product Purification kit.Use the spectrophotometry to check it.Adopt one-step RT-PCR from Ta Ka Ra company(m RNA Selective PCR Kit) to amplify them.We use Premier 5.0 Software to design all the primers.Take GAPDH as internal reference.Results: 1. The influence of indomethacin on cell viability The cells were treated with either indomethacin(final concentration 30μM) or DMSO(control) for(0,4,7) days.The result of Typan Blue Exclusion shows that cell viability of U266 and U937 cell groups decreased significantly after treated with indomethacin(Figure1-1,Figure 1-3)and it was not changed in K562 cell group(Figure1-2) treated in the same way.It prompts that this medicine(final concentration 30μM indomethacin) can inhibit the cell proliferation of U266 and U937. 2. Cell cycle analysis Compared with the control we find that both U266 and U937 cell groups were blocked at G2 / M phase after indomethacin treatment(Figure1-4,Figure1-6)and cell cyclearrest was not observed in K562 cell group checked by FCM.(Figure 1-5) 3. Apoptosis analysis The results show that cell apoptotic rates were significantly higher than their respective control groups(3.10±0.42%,1.40±0.21% and 1.38±0.10% of indomethacin treated groups vs. 0.30±0.18%,0.31±0.19% and 0.20±0.12% of control groups).It indicates that indomethacin induces the cell apoptosis of U266,K562 and U937.(Figure 1-7) 4.Cell senescence The cell senescence rate of indomethacin treated K562 and U937 cell groups increased significantly compared with their control groups(P<0.05)(Figure 1-9,Figure 1-10).The senescence rate of U266 cell group was not changed(Figure 1-8).The results indicate that this drug can induce the cell senescence of K562 and U937. 5.The expressions of p21 and p27 m RNA checked by RT-PCR Our results showed that their expression levels were significantly increased after indomethacin treatment in all three cell lines except for U937 p27 m RNA expression.(Figure 1-11, Figure 1-12)Conclutions: It is concluded that indomethacin can exert inhibitory effects on leukemia cells. However, its mechanisms were varied in different types of leukemia cells.In addition to inhibiting leukemia cell proliferation and promoting cell apoptosis, indomethacin can cause cell cycle arrest or promote cell senescence process. It prompts that non-steroidal anti-inflammatory drugs can play a role in adjuvant therapy for leukemia, while its application would be adjusted according to different types of leukemia.