| Objective: To culture DLBCL and Burkitt lymphoma cell and treat them with PMA+Iono and Z-VRPR-FMK separatily, detect cell viability, apoptosis and cell cycle, as well as MALT1 and A20 protein, investigate the effects of PMA+Iono and Z-VRPR-FMK on cell viability and the relationship between the effect and the expression of MALT1 and A20 protein, provide reliable experimental data for finding new therapeutic target for agrresive B cell lymphomas. Methods: OCI-LY1, OCI-LY8 and Raji cell were cultured, meanwhile, stimulatied with PMA+Iono and Z-VRPR-FMK; Cell viability were assayed by MTT. MALT1 and A20 protein were detected by western blotting. Apoptosis and cell cycle were examined by flow cytometry. Statistical analysis for all data was done. Results: 1. The effect of PMA+Iono and Z-VRPR-FMK on cell viability of OCI-LY8: When OCI-LY8 cell was cultured by addition of PMA(10ng/ml, 50ng/ml, 100ng/ml) for 24 h, cell vialibity had no statictical difference between experimental and control group if the concentration of Iono was lower than 1μM. P >0.05; Otherwise the OD of OCI-LY8 cell in experimental group(Adding 10ng/m, 50ng/ml, 100ng/ml PMA)(0.135±0.009, 0.126±0.019, 0.123±0.027)was less than in control group(0.173±0.032), P<0.05. When the cell was cultured and treated with 9 concentration of PMA+Iono(10ng/ml+125ng/ml, 50ng/ml+125ng/ml, 100ng/ml+125ng/ml, 10ng/ml+0.5μΜ, 50ng/ml+0.5μΜ, 100ng/ml+0.5μΜ, 10ng/ml+1μΜ, 50ng/ml+0.5μΜ, 100ng/ml+0.5μΜ) for 48 h or 72 h, The OD of cell in all experimental groups was lowered than 0.200. There was obvious difference between the experimental and corresponding control group(48h: 0.336±0.035, 72h: 0.805±0.079), P<0.05. When the cell was stimulated with PMA(10ng/ml、50ng/ml '100ng/ml) and Iono for 48 h or 72 h, there was a negative correlation between cell viability and the concentration of Iono, r and P was 48h:(-0.820,0.000),(-0.782,0.001),(-0.878,0.000);72h:(-0.800,0.000),(-0.868,0.000),(-0.919,0.000);separately; When OCI-LY8 cell was treated with Z-VRPR-FMK(25μΜ、50μΜ、75μΜ、100μΜ) for 24 h, 48 h, 72 h, The OD of OCI-LY8 cell had no changes. P>0.05. 2. The effects of PMA+Iono and Z-VRPR-FMK on MALT1 and A20 protein in OCI-LY1 and Raji cell:(1) OCI-LY1: When OCI-LY1 was cultured by addition of PMA+Iono(200ng/ml+0, 200ng/ml+125ng/ml, 200ng/ml+1μM, 200ng/ml+2μM) for 2h and PMA+Iono(200ng/ml+1μM) for 0.5h, 2h, 6h, the expression for MALT1 protein had no changes, but A20 was more in the cell treated with PMA+Iono described above than control group except the group of PMA+Iono(200ng/ml+0). For different concentration of PMA+Iono, the relative expression level of A20 was 2.44±0.77, 3.09±0.74, 4.35±0.74, 5.01±1.06 successively, P=0.118, 0.021, 0.001, 0.000. There was a positive correlation between A20 expression and the concentration of PMA+Iono, r=0.911, P =0.000. SO did between A20 expression and stimulation time of PMA+Iono, r=0.959, P=0.000, the relative expression level of A20 was 2.08±0.27 、 2.36±0.21 、 3.45±0.26 successively, P=0.001, 0.000, 0.000. When OCI-LY1 was treated with 75μM Z-VRPR-FMK for 0.25 h, 0.5h, 2h or 25μM、50μM、75μM Z-VRPR-FMK, neither MALT1 nor A20 protein changed, P>0.05; OCI-LY1 cell was cultured while it was treated with same concentration and time of PMA and different concentration and same time of Z-VRPR-FMK or invariant concentration and time of PMA and Z-VRPR-FMK for different time, MALT1 and A20 protein had no changes either, P>0.05.(2)Raji cell: Raji cell was stimulated in PMA+Iono or PMA+Iono and Z-VRPR-FMK in the same time. MALT1 protein had no significant difference, P>0.05, but A20 protein was higher in experimental than corresponding control. When Raji cell was treated with same concentration and time of PMA and different concentration and same time of Z-VRPR-FMK or invariant concentration and time of PMA and Z-VRPR-FMK for different time, A20 protein was increased with rising of concentration of Z-VRPR-FMK and stimulating time. There was positive correlation between expression for A20 and concentration of Z-VRPR-FMK, as well as the expression and stimulating time. 3. The effect of PMA+Iono and Z-VRPR-FMK on apoptosis of OCI-LY1: Apoptosis of the cell treated with PMA+Iono for 48h(25.5±8.84) was more than control one(7.43±1.42), and it was raised with stimulating time; In the cell treated with Z-VRPR-FMK for 24 h, 48 h, 72 h, the apoptosis had no increasing. 4. The effect of PMA+Iono and Z-VRPR-FMK on cell cycle: OCI-LY1 cell was cultured by adding PMA+Iono for 24 h, 48 h, the proportion of cell at S stage(19.17±3.01, 23.17±5.03, successively) was more in the treated cell than corresponding control(36.26±8.54、33.50±5.10), P <0.05. When Raji cell was treated with described above for 72 h, the proportion of cell at G2-M stage was more in treated cell(7.05±1.75) than control cell(2.65±0.83), P <0.05. Conclusion: 1. PMA+Iono can suppresses viability of DLBCL, it may be associated with the expression of A20 protein. 2. MALT1 protease activity of MALT1 is increasing in Burkitt lymphoma cell, MALT1 may be a new therapeutic target for Burkitt lymphoma. 3. There is no increasing of MALT1 protease activity of MALT1. PMA+Iono may be associated with increased A20 expression in GCB-like DLBCL. |
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