Mechanisms Of Chidamide In Diffuse Large B-cell Lymphoma Cell Lines With Distinctive Proliferative Characteristics

BackgroundDiffuse large B-cell lymphoma is the most common subtype of malignant lymphoma, accounting for 30%-40% of all non-Hodgkin’s lymphoma in adult,showed significant heterogeneity in morphology,cytogenetics,immune phenotype,clinical presentation,response to chemotherapy and prognosis.Rituximab appears to be considered as a major breakthrough in the treatment of B-cell lymphoma, which significantly improved the response rate and overall survival of the patients.However, there are still more than 30% of the DLBCL patients presence of primary or secondary chemoresistance to Rituximab immuno-chemotherapy,these considerations have promoted the search for more effective strategies in DLBCL to improve prognosis.Epigenetic aberrations,which are recognized as key drivers of human cancer,have intimate connections with the occurance and progression of many tumors.Studies found that the imbalance of histone acetyltransferases and histone deacetylases can inhibit gene expression,including those that regulate cell cycle,cell apoptosis,cell differentiation and tumor immune,resulting in the development of tumors. Inactivating mutations in the histone acetyltransferases,CREBBP and EP300 were found to occur in approximately 30% of DLBCL patients, which have intimate connections with the occurance and progression of DLBCL.Histone deacetylase inhibitors can induce tumor cell growth arrest, apoptosis and cell cycle arrest by promote histone acetylation,showed a better efficacy in human malignancies. Chidamide is a new generation of amides HDACis designd and manufactured in China independently,selectively inhibit the expression of HDAC1,2,3,10,and it has been approved for the treatment of relapsed or refractory peripheral T cell lymphoma,also shown certain effect in both leukemia and colorectal malignant tumor and other malignant tumors.But its effects for DLBCL have not been clear yet. This experiment will study the effect of Chidamide on DLBCL cell lines,explore its possible mechanisms and whether Chidamide can use for the treatment of DLBCL,searching new methods for increasing clinical therapeutic effect of Chidamide on DLBCL. ObjectiveTo explore the effect of Chidamide on DLBCL cell lines of apoptosis、cell cycle、cell differentiation and its possible mechanisms, in order to find new approachs to increasing clinical therapeutic effect of Chidamide on DLBCL. Materials and Methods1. Cell culture and subculture:Select three kinds of DLBCL cell lines with different proliferative characteristics, OCI-Ly3, OCI-Ly7 and OCI-Ly8 to culture and passage.2. To detect the apoptosis rate of OCI-Ly3, OCI-Ly7 and OCI-Ly8 cell lines treated with different drugs and concentrations by Annexinv-FITC/PI double staining flow cytometry.3. To detect the cell cycle of OCI-Ly3, OCI-Ly7 and OCI-Ly8 cell lines treated with different concentrations of Chidamide by flow cytometry.4. To detect the expression of CD20, CD38, CD138, H3 and ac-H3 by Western blot.6. Use SPSS13.0 software for statistical analysis, P<0.05 was considered statistically significance. Results1. The proliferative characteristics of OCI-Ly3、OCI-Ly7 and OCI-Ly8 cell lines are different:Cell cycle results suggest that the proportion of G0/G1 phase of the three DLBCL cell lines are 30.23%、27.99% and 30.45% respectively, the proportion of S phase of the three DLBCL cell lines are 39.86%、70.23% and 54.46% respectively, and the proportion of G2/M phase of of the three DLBCL cell lines are 27.80%、2.51% and 11.64% respectively.The S phase and G2/M phase of the three DLBCL cell lines were significantly different(P<0.001).2.Chidamide induced OCI-Ly3、OCI-Ly7 and OCI-Ly8 cell lines apoptosis to various degrees: Cell apoptosis rate increased with the growing concentration of Chidamide,but to cell lines with different proliferative characteristics,the ability of Chidamide to induce apoptosis was significantly different(P=0.0020).OCI-Ly7 cell line had the most obvious apoptosis-inducing effects followed by OCI-Ly8 and OCI-Ly3 cell lines. For OCI-Ly7 cell line, the apoptosis rate ranged from 21.07% to 96.54%. For OCI-Ly8 cell line,it ranged from 4.56% to 40.98%, and for OCI-Ly3 cell line, it ranged from 5.84% to 12.83%.3. Chidamide induced a G0/G1 cell cycle arrest in OCI-Ly3、OCI-Ly7 and OCI-Ly8 cell lines to various degrees:The proportion of G0/G1 phase of the three DLBCL celllines increased with the growing concentration of Chidamide(P=0.0388), together with the S phase proportion down-regulated(P=0.0015),but it has little effect on G2/M phase(P>0.05).OCI-Ly3 cell line had the most significant effect treated with Chidamide followed by the OCI-Ly8 and OCI-Ly7,the inhibiting effect increased significantly with the growing concentration of Chidamide, and the proportion of G0/G1 phase rised from 30.23% to 74.40%.For OCI-Ly8 cell line, the proportion of G0/G1 phase rised from 30.45% to 68.81%. For OCI-Ly7 cell line, the proportion of G0/G1 phase rised from 27.99% to 38.47%.4. Chidamide induced cell differentiation in OCI-Ly3、OCI-Ly7 and OCI-Ly8 cell lines to various degrees: Chidamide increased the expression of CD20 and CD38 protein in OCI-Ly7 cell line, without affecting the expression of CD138 protein, and the expression of CD20 protein increased significantly with the lapse of time; In OCI-Ly3 cell line, the expression of CD20 protein increased slightly, without affecting the expression of CD38 and CD138 protein; Chidamide had no effect on the expression of CD20、CD38 and CD138 protein of OCI-Ly8 cell line.5. Chidamide influenced the expression of ac-H3 protein varying degrees in OCI-Ly3 、 OCI-Ly7 and OCI-Ly8 cell lines: Chidamide had no effect on the expression of H3 protein, but affected the expression of ac-H3 protein in different degrees. The expression of ac-H3 protein in OCI-Ly7 cell line increased continuously with time going on. For OCI-Ly8 cell line, the expression of ac-H3 protein declined slightly with time going on; For OCI-Ly3 cell line, as time going on,the expression of ac-H3 protein decreased significantly.6. The combination of Chidamide and other chemotherapeutic agents improved the efficacy of Chidamide alone. The combination of Chidamide and Rituximab significantly increased the apoptosis rate of OCI-Ly7 cell line(P<0.05), but didn’t increase the apoptosis rate of OCI-Ly3 and OCI-Ly8 cell lines(P>0.05).The combination of Chidamide and Doxorubicin,as well as the combination of Chidamide and Decitabin increased cell apoptosis rate of all the three DLBCL cell lines significantly(P<0.05). Conclusions1. Chidamide can induce the DLBCL cell lines apoptosis、arrest the cell cycle at G0/G1, and induce cell differentiation,but the effects on cell lines with different proliferative characteristics are disparity.2. The effects of Chidamide on cell lines with different proliferative characteristics are related to the level of acetylation induced by Chidamide.3. The combination of Chidamide and other drugs can improve therapeutic effect,the combination of Chidamide and Doxorubicin,as well as the combination of Chidamide and Decitabin are effective to the all the DLBCL cell lines with different proliferative characteristics.