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Experimental Research of Cell Proliferation and Apoptosis of All-trans Retinoic Acid Combined with Temozolomide on U251Glioma Cell Line

Author: WuChangSong
Tutor: XuGuoZheng
School: Southern Medical University,
Course: Surgery
Keywords: All-trans retinoic acid Temozolomide U251glioma cell line Proliferation inhibition Apoptosis Sensitization
CLC: R73-36
Type: Master's thesis
Year: 2013
Downloads: 4
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Background:Chemotherapy is one of the important adjuvant therapies for the treatment of glioma, but the types of glioma chemotherapeutic drugs are rare. Oral alkylating agent temozolomide (TMZ), which is biocompatible and able to penetrate the blood-brain barrier, is the most common anti-glioma agent and the preferred standard chemotherapy for glioma. After absorbed into the body,TMZ can be decomposed to5-amino-imidazole-4-amide (AIC) and diazomethane.The later is the active alkylating substances, which produce cytotoxic effects, and methylate the N7and O6of guanine in the cell’s DNA so as to disable cell’s mismatch repair system finding the matching bases for the O6of the methylated guanin in the process of DNA replication, and finally result in gaps in the DNA sub-chain. The gaps accumulate more and more in the process of cell division, and eventually hindering the start of DNA replication, so that the cell cycle arrest at the G2/M phase and cells apoptosis are induced. However, this effect will be reversed by resistance-associated protein O6-methylguanine-DNA-methyltransferase (MGMT). MGMT can repair DNA alkylation damage induced by alkylating agent, which is the main reason for the drug resistance of nitrosourea drugs and TMZ, thus limit the anti-tumor effect of TMZ. How to apply a chemotherapy sensitizer to improve TMZ cytotoxicity for malignant glioma is a research hotspot in recent years. All-trans retinoic acid (ATRA), a natural inducer, can induce cell differentiation, and inhibit cell proliferation and induce apoptosis of tumor cells, therefore it have anti-tumor effect that have been proved in the treatment of the hematopoietic system tumors and other solid malignances (such as liver cancer, lung cancer, colorectal cancer and breast cancer). Especially, ATRA can induce complete remission of acute promyelocytic leukemia. Some studies have shown that ATRA inhibit the proliferation of C6glioma cells and the inhibition rate is positive correlation with ATRA concentration and treatment time. As a fat-soluble compound, ATRA can penetrate the blood-brain barrier, and concentrate in the brain, which is the basis for the treatment of glioma. Currently, the mechanism of retinoic acid on glioma is not completely clear. It is involved in the entire network system of proliferation, differentiation and apoptosis regulation. There is not much report about ATRA in the treatment of glioma, and the anti-tumor effect of it when singly used is not very good, but it has been reported that through its ability of inducting cell differentiation and inhibiting cell proliferation, ATRA could increase sensitivity of glioma cytotoxicity of other chemotherapy drugs such as paclitaxel, daunorubicin and y-interferon. In this experiment, we used U251human glioma cell line as an experimental model, and used ATRA as a sensitizer for TMZ anti-tumor effect to observe whether there are synergies for cells growth inhibition, whether ATRA can sensitize the effect of inducing cell apoptosis of TMZ when ATRA and TMZ were combined used on U251cells, and to preliminarily analyze its possible mechanism by detecting related regulatory proteins of cell differentiation, apoptosis and the expression of cell phenotype molecular(such as CD133phenotype, apoptotic protein of Bcl-2, BAX, caspase-3, caspase-8, caspase-9), so as to provide an experimental basis for further research of overcoming TMZ resistance of glioma cells and enhancing TMZ anti-glioma effect in vitro.Methods:1Cell culture:U251glioma cell line was cultured in DMEM/F12medium containing10%fetal bovine serum (FBS) in37℃,5%CO2incubator. The culture medium was changed every1-2days and the cells were passaged every4-5days. We took U251cells in logarithmic growth phase for experiments.2Detection of growth inhibition rate of U251cells by MTT method:U251cells in logarithmic growth phase were digested and suspended, then the cells were counted and the cell concentration was adjusted, followed by inculation into96-well plates, with each well added100μl culture medium containing5×103cells. There were three groups: control group, ATRA group and TMZ group. We set concentration of10,20,30,40mM for ATRA, and100,200,300,400mM for TMZ, each drug concentration group and control group contained4wells. We planted5plates in each experiment, and cultured them in37℃,5%CO2incubator respectively after24,48,72,96and120h. The supernatant was removed and appropriate concentrations of drugs were added. The control group was treated by the corresponding concentration of dimethyl sulfoxide (DMSO). Each plate was cultured in37℃,5%CO2incubator for another24h, and then underwent MTT colorimetric experiments.2μl MTT (5g/L) was added into each well and the plates were incubated for4h, and the supernatant was absorbed carefully, and150μl DMSO was added in each well. These plates were put on shaking table for10min, and then microplate reader was used to measure the OD values at490nm, draw growth curves and make statistical analysis. According to the experimental results, we chose ATRA concentration of20mM treating the cells for48h and removed supernatant followed by the treatment of200mM of TMZ for another48h, then measured cell growth inhibition rate by MTT assay and compared the inhibition rate of the two drugs at the same concentration after48h. The cell growth inhibition rate=(1-OD value of experimental group/OD value of the control group)×100%.3Grouping and drug intervention:Blank control group (the same amount of DMEM/F12medium plus the same amount of DMSO), ATRA group (20mM), TMZ group(200mM), and combination group (ATRA concentration of20mM+TMZ concentration of200mM). Two drug groups were treated respectively according to the above grouping for48hours; the combination group was treated in order:firstly ATRA (20mM) was used for48h, then culture medium was absorbed and TMZ (200mM) was used for another48h.4Scratch test and observation of changes in cell morphology:Each group was treated by the drug concentration and method mentioned above and cultured in the condition of37℃,5%CO2. At12,24,48h we observed the cell morphological changes using an inverted phase contrast microscope. The logarithmic growth phase cells were inculated into6-well plates with a density of1.0±106/ml and cultured for24h in DMEM/F12containing10%FBS until the cells covered more than80%of the board. Then10μl pipette tips were used to scratch a straight line in the middle of each well which were then washed three times with sterile PBS to remove suspended cells, drugs were added and cells were cultured according to grouping mentioned above. Then the cell migration was observed and the pictures were taken using an inverted phase contrast microscope.5Detection of cell CD133positive rate by immunofluorescence:The logarithmic phase U251cells were inoculated in6-well plate in which a glass slide was placed in each well in order to let cells grow on the slides, and2ml DMEM/F12medium containing10%FBS was added in each well. When the cells grew well, they were treated according to the grouping. The glass slides were taken out, washed by PBS for three times, fixed in paraformaldehyde for10minutes, and then washed three times with PBS for10minutes each to remove the paraformaldehyde, and immersed into0.5%Triton-X100for10minutes. Then the slides were washed again and blocked with1:10normal goat serum diluted by PBS for10minutes at room temperature. We removed liquid, and added0.01M appropriate dilution of rabbit anti-human CD133monoclonal antibody overnight at4℃. The slides were washed with PBS for2minutes X3times, incubated at37℃for30min with FITC-labled secondary anti-rabbit IgG antibody, rinsed with PBS for5min×4times, blocked with water-soluble mounting medium, and observed under fluorescent microscope. Excitation with495nm fluorescence, the positive cells would produce green fluorescence. The rate of CD133positive cells=CD133positive cells/adherent cells×100%.6Detection of apoptotic rate of U251cells by flow cytometry:logarithmic growth phase cells were digested and inoculated into6-well plates. After cells adhered on the plates for24h, the drugs were added according to the grouping. The cell morphologic and histological changes were investigated under the inverted phase contrast microscope. Taking DMEM/F12medium as a blank control, U251cells were digested and suspended, and then washed and centrifugated twice. The U251cells were resuspended with500μl of binding buffer, followed by adding5μl AnnexinV-FITC and5μl propidium iodide (PI) staining solution. Then we mixed and placed them into ice bath and dark place for15min. Flow cytometry was used to detect the apoptotic rate of U251cells in each group. The data was analyzed using Cell Quest software. Apoptotic cells were characterized by Annexin V-FITC+PI+and Annexin V-FITC+PI-.Apoptotic rate was calculated after10000cells were counted.7Cell protein expression detected by western blot assay:U251cells were treated according to the grouping mentioned above. We rinsed U251cells twice with PBS, added the pre-cooling total cellular protein extraction reagent for20min, and centrifugated twice with12000r/min at4℃. The supernatant was extracted and Bradford method was used to measure protein concentration. We added1/4volume of SDS-PAGE buffer into cell lysis buffer, then loaded40μg sample per lane on electrophoresis apparatus, followed by separating protein and transferring them to PVDF membranes. We blocked them by5%skimmed milk at room temperature for30min and treated the cells by the first antibody of Bcl-2(1:1500), Bax (1:1000), caspase-3(1:500) of caspase-8(1:1000), caspase-9(1:500) and (3-Actin (1:1000) at room temperature for2h, rinsed them by PBST for3times. HRP labeled anti-rabbit IgG antibody (1:2000) was added and the membranes were incubated at room temperature for45min. The membranes were washed by PBST and developed by ECL method. β-Actin (1:1000) was taken for internal reference. The data was analyzed by AlphaEase FC software to determine the gray level of each protein in each group, and the relative content of protein was calculated by the ratio of grey level of target protein and the one of the reference.8Statistical Methods:Data was analyzed by SPSS13.0statistical software, and the results were presented as mean±standard deviation (X±s). Single factor variance analysis was used to analyze the data among multi-groups, and the significance between two specific groups was detected with q-test. Statistic difference was considered to be significant if P<0.05.Results:1Inhibition of cell proliferation There were no cell proliferation inhibition produced by10mM ATRA treated for24h. On the contrary, it can promote cell proliferation to some extent, showing increase in the absorbance, and the inhibition rate was (-13.35±6.84)%. After48h it started to inhibit cell proliferation, but the inhibition rate is low (13.18±9.46)%at48h. At ATRA concentration of20,30,40mM, the inhibition rate increased with the increase of drug concentration and the prolongation of action time, and the fastest increase occurred at48h, followed by a gentle elevation of growth inhibition rate at72,96and120h. Among these drug concentrations,20mM ATRA achieved the fastest growth inhibition rate, amounted to (43.64±5.09)%, and there were statistically significant differences (P<0.01) compared with10mM group. The inhibition rate of TMZ increased with its increase of concentration and action time; the inhibition rate was low at the concentration of100mM which was only (23.37±8.19)%at48h, while at200mM the inhibition rate was (35.77±5.39)%and there was statistically significant difference (P<0.01). At72,96and120h the elevation of inhibition rate became significantly slow in both ATRA and TMZ groups, and there were no statistically significant differences between the three time points (P>0.05). We could draw proliferation inhibition curves of the two drugs according to these statistical data. We saw that using20mM ATRA and200mM TMZ for48h significantly inhibited cell proliferation, so we selected the ATRA concentration of20mM and the TMZ concentration of200mM to be the experimental does for each single used group and the combination group. In the combination group, the cell proliferation inhibition rate detected by MTT assay was (76.33±5.65)%afer treated by20mM ATRA for48h followed by the treatment of200mM TMZ for another48h, and there were statistically significant differences (P<0.01) compared with ATRA group and TMZ group, while there was no statistically significant difference (P>0.05) between ATRA group and TMZ group.2The morphologic changes of U251cells observed by inverted phase contrast microscope:U251cells in control group were irregular fusiform and had much synapses, good diopter, abundant cytoplasm and clear edge, and bonded to the culture plate closely. After treated by ATRA and TMZ for12,24and48h, majority of cells became round and small, and the diopter was low, and some cells floated. As the time went on, the changes of U251cells became more significant. Majority of U251cells treated by the combination group for96h were in round and small shape. They were separated from the plates and floated in the culture medium, with vague margin and decreased diopter, and fragments of apoptotic cells can be observed. In scratch test, each group had a same width of cell-free scratch area. When treated according to each group, the migration ability of U251cells in control group was normal and the cells nearly covered the scratch area. The cell migration of ATRA and TMZ groups were visible and the scratch areas were narrowed, but the migration ability was weak and the cells only partially covered the scratch area. In the combination group, U251cells showed no migration and the scratch area did not change compared with the one before treatment.3CD133positive rate detected by immunofluorescence:Under immunofluorescence microscope, FITC-labled secondary antibody showed bright green excited by495nm fluorescence. The cell markers of CD133+were labled on the cell membrane. The cells were in round or nearly-round shape, and it was easy to cluster when the cells were in larger quantities. When treated according to each group, the positive rate of CD133cells in control group was (7.05±0.27)%. In ATRA group it significantly decreased to (0.66±0.30)%, while the highest positive rate was up to (36.32±3.22)%in TMZ group. In the combination group, it fell to (4.70±0.52)%. There were statistically significant differences(P<0.01) among the groups.4Apoptosis of U251cells induced by ATRA and TMZ:The apoptotic rate of U251cells were detected by flow cytometry after treated by20mM ATRA and200mM TMZ for48h. Both drugs could induce apoptosis in U251cells, and the apoptosis rates were respectively (24.93±4.58)%and (21.36±3.76)%, while in combination group the apoptosis was significantly enhanced and the apoptosis rate was (41.66±5.02)%. The apoptosis rate in ATRA and TMZ groups were both significantly higher than that in the control group (P<0.01), and in the combination group it was significantly higher than that in the single drug groups and the control group (P <0.01). This demonstrated the fact that ATRA could significantly enhance the effect of inducing apoptosis of TMZ in U251cells. There was no statistically significant difference (P=0.05) between the two single drug groups.5Protein expression of Bcl-2, BAX, caspase-3, caspase-8and caspase-9in U251cell detected by Western blot:experiment results demonstrated that to varying degrees there were high expressions of BAX, caspase-3, caspase-8and caspase-9in each interference group, and the highest expression was in the combination group. In contrast, the expression of Bcl-2was low in each interference group, and the lowest expression was in the combination group. There was statistically statistical significance (P<0.01) between each treatment group and control group, and between the combination group and the two drug-alone groups. The expression of Bcl-2between ATRA group and TMZ group had no statistically statistical significance (P>0.05), while the expression of Bax, caspase-3, caspase-8and caspase-9in ATRA group were higher than that in TMZ group and there was statistically statistical significance (P<0.05).Conclusions:1ATRA and TMZ can significantly inhibit the proliferation and migration of U25glioma cell line in vitro, and the effects are significantly strengthened by the combination use of the two agents.2The inhibition effect of both ATRA and TMZ for U251cell line is dose-and-time-dependent, which means the inhibition effect ascends with the increase of the concentration of both drugs and with the prolongation of treatment time.3ATRA and TMZ can significantly induce apoptosis in U2S1cells, and with the sequential application of ATRA and TMZ, the induction of apoptosis can be significantly enhanced, which means that the combination use of the two drugs has a synergistic effect and proves that ATRA can sensitize the cytotoxicity of TMZ.4The possible mechanisms of inhibiting proliferation and promoting apoptosis of ATRA and TMZ in U251cells may associate with down-regulating protein expression of Bcl-2and up-regulating the protein expression of Bax, caspase-3, caspase-8and caspase-9in U251cells. The chemotherapeutic sensitization of ATRA to TMZ may due to the reason that ATRA can induce differentiation of CD133positive cells and reduce their quantity in U2S1cell line.

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