Expression of enhancer of zeste homolog 2 in U266 cell line and bone marrow mononuclear cells of multiple myeloma patients / 白血病·淋巴瘤

Objective To investigate the expression of DNA methylation related protein enhancer of zeste homolog 2 (EZH2) in multiple myeloma (MM) cell line U266 and enriched MM cells (MM-BMMNC) from 10 patients,and analyze the potential role of DNA methylation in the occurrence and development of MM.Methods Human MM cell line U266 and MM cells from 10 MM patients were studied.Enriched bone marrow mononuclear cells (N-BMMNC) from 10 healthy persons were used as the control.The expression of EZH2 were determined by Western blot using the ratio of target protein EZH2 and the internal reference β-actin expression as the final results.Results The preliminary in vitro results showed that EZH2 expression was higher in MM cell line U266 and patient MM-BMMNC (N-BMMNC 0.2277±0.1306,MM-BMMNC 0.9220±0.1301,U266 0.9730±0.1029),with statistically significant differences (P ＜ 0.05).Conclusion EZH2 is enriched in U266 and MM-BMMNC.DNA methylation may have a close correlation with the occurrence and development of MM.

Study on histone deacetylase inhibitor LBH589 reversal of multiple myeloma cells’ drug resistance mechanism in vitro / 白血病·淋巴瘤

Objective To study on a new generation of histone deacetylase inhibitor LBH589 on multiple myeloma (MM) resistant cells associated with changes in gene expression,and the mechanism of LBH589 reversal of MM cells' drug resistance.Methods By Western blot analysis,LBH589 (0,20,50 nmol/L) and 50 nmol/L LBH589 combined respectively with dexamethasone of 5 μmol/L at 24 h,expression of histone H4 acetylation and bcl-X gene on MMIR cells were detected.By real-time fluorescence quantitative PCR analysis,LBH589 (0,20,50 nmol/L) and 50 nmol/L LBH589 combined respectively with dexamethasone of 5 μmol/L at 24 h and 48 h,expression of TOSO on MM1R cells were detected.Results Western blot analysis showed that single LBH589 and combined with dexamethasone showed at 24 h the up-regulation on expression of the histone H4 acetylation[(0.205±0.008) ％,(0.346±0.009) ％,(0.580±0.053) ％,(0.986±0.012) ％,F =992.957,P =0.032],while down-regulation on expression of the bcl-X in MM1R cells in a dose-dependent manner[(1.210±0.160) ％,(0.930±0.036) ％,(0.730±0.017) ％,(0.488±0.029) ％,F =56.432,P =0.028].However,the group of single LBH589 was less effective than the combined group (all P ＜ 0.05).Real-time fluorescence quantitative PCR analysis showed single LBH589 and that combined with dexamethasone showed at 24 h and 48 h,the down-regulation on expression of the TOSO in MM1R cells in a dose-dependent manner,24 h specific numerical were (1.00±0.00) ％,(0.55±0.01) ％,(0.47±0.01) ％,(0.38±0.01) ％ (F =1006.344,P =0.041),48 h specific numerical were (1.00±0.00) ％,(0.39±0.04) ％,(0.05±0.01) ％,(0.03±0.00) ％ (F =2383.977,P =0.045),however,the group of single LBH589 was less effective than the combined group (all P ＜ 0.05).Conclusion Histone deacetylase inhibitor LBH589 can recover dexamethasone sensitivity of MM1R through effect the gene expression of bcl-X and TOSO,promot histone acetylation,and inducing cell apoptosis to treat tumor.

Inhibitory effect of histone deacetylase inhibitor LBH589 on multiple myeloma MM1R cells in vitro / 中国实验血液学杂志

This study was purposed to explore the effect of a new generation of histone deacetylase inhibitor LBH589 alone or combined with bortezomib (Bor) on multiple myeloma cells (MM1R) in vitro. The effect of LBH589 (10, 20, 50 nmol/L) alone or combined with Bor (10, 20 nmol/L) on MM1R proliferation was detected by MTT method; the effect of LBH589 on cell cycle and apoptosis of MM1R cells were determined by flow cytometry; the histone H4 acetylation level of MM1R cells treated with LBH589 (10, 20, 50 nmol/L) for 24 h was analyzed by Western blot. The results showed that the LBH589 alone or combined with Bor all could inhibit the proliferation of MM1R cells in a concentration- and time-dependent manner. After MM1R cells were treated with drugs for 48 h, the cells in G(0)/G(1) phase increased, the cells in G(2)/M and S phase decreased, suggesting the arrest of cells in G(0)/G(1) phase, at the same time, the apoptosis rate of MM1R cells treated with drugs increased in a concentration-dependent manner, while the effect of LBH589 combined with Bor was more obvious than that of LBH589 alone (P < 0.001). Western blot analysis showed that the histone H4 acetylation level was enhanced in concentration-dependent manner after MM1R cells were treated with different concentrations of LBH589 for 24 h. It is concluded that the LBH589 can inhibit the proliferation of MM1R cells, block the cell cycle, induce cell apoptosis, moreover LBH589 combined with Bor has synergistic effect on MM1R cells.

Silencing of ICSBP/IRF8 expression in U226 cells and bone marrow mononuclear cells from patients with multiple myeloma / 中国实验血液学杂志

The objective of this study was to investigate expression of interferon regulatory factors (ICSBP/IRF8) and the potential role of DNA methylation in silencing ICSBP/IRF8 gene in multiple myeloma (MM) cell line U266 and bone marrow mononuclear cells from 10 MM patients (MM-BMMNC). The bone marrow mononuclear cells from 10 healthy persons (N-BMMNC) were collected and used as normal controls. Expression of ICSBP/IRF8 gene was detected by real-time fluorescence quantitative PCR (using 2(-ΔΔCT) to calculate); DNA methylation level of the ICSBP/IRF8 gene was measured using methylation-specific PCR (using the ratio of interest gene ICSBP/IRF8 and internal reference β-actin expression as results). The results showed that as compared with N-BMMNC the lower expression of ICSBP/IRF8 gene was found in U266 cells and MM-BMMNC, the hypermethylation of the CpG island in the ICSBP/IRF8 promoter was observed, there were significant differences between N-BMMNC and MM-BMMNC or U266 cells (P < 0.05). It is concluded that the expression of ICSBP/IRF8 gene can be silenced in the MM-BMMNC and U226 cells. As the hypermethylation of CpG island in ICSBP/IRF8 promoter is a frequent event in MM cells, the ICSBP/IRF8 gene silencing caused by DNA methylation may take part in the pathogenesis and development of MM.

Study on histone deacetylase inhibitor LBH589 induces apoptosis of multiple myeloma cells and its reversal of drug resistance mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact of a new generation of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on multiple myeloma (MM) cells proliferation and its mechanism.</p><p><b>METHODS</b>MM cell line U266 and dexamethasone resistant cell line MM1R cells were treated with different concentrations of LBH589 alone or in combination with bortezomib, the inhibition of cells proliferation was detected by MTT, the cell cycle and apoptosis by flow cytometry. The expression level of histone H4 acetylation and PARP, Bcl-X protein was analyzed by western blot, expression level of caspase-3, APAF-1 and TOSO gene by real-time fluorescence quantitative PCR.</p><p><b>RESULTS</b>U266 and MM1R cell proliferation were inhibited by different concentrations of LBH589 (0, 10, 20, 50 nmol/L) alone or 50 nmol/L of LBH589 in combination with bortezomib (10, 20 nmol/L) in a dose- and time-dependent manner. Inhibition effect was significantly higher in all combinative groups than in single agent groups (all P < 0.05). The percentage of G(0)/G(1) phase in MM1R cells were 36.60%, 46.50%, 51.40%, 57.10%, 75.48%, 79.73%, respectively, and the apoptosis rate were 5.27%, 31.41%, 39.78%, 44.07%, 73.60%, 83.27%, respectively. The effects appeared to occur in a dose-dependent manner, and being significantly higher in all combinative groups than in single agent groups (all P < 0.05). The expression of the caspase-3 and APAF-1 gene up-regulated gradually, while TOSO gene expression in MM1R cells down-regulated gradually in a dose- and time-dependent manner (all P < 0.05).</p><p><b>CONCLUSIONS</b>LBH589 can inhibit the growth of MM cells, block the cell cycle and induce cell apoptosis, which has anti-resistant effect on multidrug resistant cell. At the same time LBH589 in combination with bortezomib on myeloma cell has a synergistic effect, its mechanism and reversal of drug resistance mechanism involves in multiple changes in gene expression.</p>