Effect of decitabine combined with adriamycin on TP53 mutated diffuse large B lymphoma cell line and its clinical significance
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摘要: 目的 观察地西他滨对TP53突变弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma，DLBCL)细胞株DB增殖、凋亡及周期的影响，并评估地西他滨联合治疗TP53突变DLBCL患者的疗效及安全性。方法 体外培养细胞，不同浓度(5~80 μmol/L)地西他滨和阿霉素作用于DB细胞后采用CCK-8法计算增殖抑制率；地西他滨(20 μmol/L)和阿霉素(12 μmol/L)作用于DB细胞24/48 h，采用Annexin V-FITC/PI及流式细胞术检测凋亡率及细胞周期；比较不同组间细胞增殖、凋亡及周期的差异。另收集5例地西他滨联合治疗TP53突变DLBCL患者的临床资料进行回顾性总结。结果 对DB细胞的增殖抑制作用随着地西他滨浓度的增加(5、10、20、40、80 μmol/L)而增强，联合阿霉素(12 μmol/L)后抑制率较单药明显增大[(78.51±1.19)% vs (40.80±1.62)%，(87.48±0.29)% vs (46.83±1.47)%，(92.59±0.15)% vs (47.07±1.50)%，(94.57±0.58)% vs (52.68±0.84)%，(95.56±0.53)% vs (54.95±1.07)%，P < 0.01]；采用IC50浓度(20 μmol/L地西他滨、12 μmol/L阿霉素)药物处理细胞，抑制率随时间增加(12、24、48 h)而增大[(50.40±0.19)%、(61.65±0.16)%、(85.08±0.78)%，P < 0.01]。与空白对照组比较，地西他滨作用于DB细胞24 h显示S期细胞比例增加[(10.54±0.40)% vs (2.03±0.01)%，P < 0.01]，48 h显示G2/M期细胞比例增加[(17.45±0.19)% vs (3.69±0.09)%，P < 0.01]。5例复发/难治TP53突变DLBCL患者使用含有地西他滨方案治疗后，总有效率为40%(2/5)，主要不良反应为骨髓抑制，未出现治疗相关死亡。结论 地西他滨可增强阿霉素对TP53突变DLBCL细胞株的增殖抑制作用，阻滞细胞周期。含地西他滨方案治疗TP53突变DLBCL显示出一定疗效，安全性良好，值得进一步探索。Abstract: Objective To observe the effect of decitabine(DAC) on the proliferation, apoptosis and cell cycle of TP53 mutated diffuse large B cell lymphoma(DLBCL) cell line DB, and evaluate the efficacy and safety of DAC combined therapy in patients with TP53 mutant DLBCL.Methods Cells were cultured in vitro. The different concentrations(5-80 μmol/L) of DAC and adriamycin(ADM) act on DB cells and the CCK-8 method was used to calculate the proliferation inhibition rate. DAC(20 μmol/L) and ADM(12 μmol/L) act on DB cells 24, 48 h, Annexin V-FITC/PI and flow cytometry were used to detect the apoptosis rate and cell cycle. The differences in cell proliferation, apoptosis and cell cycle in different groups were compared. Clinical data of 5 patients with TP53 mutated DLBCL treated with DAC combination was retrospectively collected and summarized.Results The inhibitory effect on the proliferation of DB cells increased with the increase of DAC concentration(5, 10, 20, 40, 80 μmol/L), and the inhibition rate in combination with ADM(12 μmol/L) was significantly higher than that of single drug ([78.51±1.19]% vs [40.80±1.62]%, [87.48±0.29]% vs [46.83±1.47]%, [92.59±0.15]% vs [47.07±1.50]%, [94.57±0.58]% vs [52.68±0.84]%, [95.56±0.53]% vs [54.95±1.07]%, P < 0.01). When the cells were treated with IC50(20 μmol/L DAC, 12 μmol/L ADM), the inhibition rate increased ([50.40±0.19]%, [61.65±0.16]%, [85.08±0.78]%) with the increase of time(12, 24, 48 h)(P < 0.01). Compared with the blank control group, the percentage of DB cells in S phase increased after treatment with DAC for 24 h ([10.54±0.40]% vs [2.03±0.01]%, P < 0.01), and the proportion of cells in G2/M phase increased at 48 h ([17.45±0.19]% vs [3.69±0.09]%, P < 0.01). The five patients with recurrent refractory TP53 mutation DLBCL treated with DAC regimen, the total effective rate was 40%(2/5). The main adverse event was myelosuppression and there was no treatment-related death.Conclusion DAC can enhance the inhibitory effect of ADM on the proliferation of TP53 mutant DLBCL cell line, and block cell cycle. The regimen containing DAC shows a certain efficacy and safety in the treatment of TP53 mutation DLBCL, which is worthy of further exploration.
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Key words:
- diffuse large B cell lymphoma /
- relapsed or refractory /
- TP53 mutation /
- adriamycin /
- decitabine
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表 1 药物作用48 h对DB细胞的抑制作用
X±S 添加药物 抑制率/% 5 μmol/L 10 μmol/L 20 μmol/L 40 μmol/L 80 μmol/L ADM 40.78±1.25 47.77±1.06 62.68±0.47 74.43±1.91 79.71±1.82 DAC 40.80±1.62 46.83±1.47 47.07±1.50 52.68±0.84 54.95±1.07 ADM(12 μmol/L)+DAC 78.51±1.19 87.48±0.29 92.59±0.15 94.57±0.58 95.56±0.53 P < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 
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表 2 药物作用不同时间对DB细胞的抑制作用
X±S 添加药物 抑制率/% 12 h 24 h 48 h P△ 12 μmol/L ADM 37.66±0.50 45.04±0.19 56.85±0.57 < 0.001 20 μmol/L DAC 32.85±0.21 40.50±0.16 45.49±1.84 < 0.001 两药联合 50.40±0.19 61.65±0.16 85.08±0.78 < 0.001 P▲ < 0.001 < 0.001 < 0.001 △12 μmol/L ADM、20 μmol/L DAC和两药联合在不同时间对DB细胞抑制率的比较；▲在相同时间内12 μmol/L ADM、20 μmol/L DAC和两药联合对DB细胞抑制率的比较。
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表 3 药物作用24 h对DB细胞周期的影响
%，X±S 组别 G0/G1 S G2/M 空白对照组 90.38±0.19 2.03±0.01 3.38±0.10 12 μmol/L ADM 82.29±0.66 12.56±0.71 4.78±0.33 20 μmol/L DAC 86.78±0.48 10.54±0.40 2.17±0.08 两药联合 79.37±0.51 16.55±0.73 3.46±0.13 P < 0.001 < 0.001 < 0.001
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表 4 药物作用48 h对DB细胞周期的影响
%，X±S G0/G1 S G2/M 空白对照组 90.35±0.17 3.89±0.16 3.69±0.09 12 μmol/L ADM 75.94±0.51 4.01±0.06 19.39±0.27 20 μmol/L DAC 78.90±0.42 4.46±0.18 17.45±0.19 两药联合 75.00±0.35 1.85±0.09 24.91±0.40 P < 0.001 < 0.001 < 0.001
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表 5 患者临床资料
病例 性别 年龄/岁 分型 IPI评分 ECOG评分 TP53突变类型 既往治疗线数 治疗方案 疗效 不良反应 1 男 58 GCB 3 2 exon 4 2 DAC、PD-1 疾病进展 Ⅳ度骨髓抑制、肝损害 2 女 37 non-GCB双表达 4 3 无 3 DAC、PD-1 疾病稳定 Ⅳ度骨髓抑制、严重腹泻 3 女 55 GCB双表达 2 1 exon 7 2 DAC、PD-1、泽布替尼 2个疗程部分缓解，4个疗程疾病稳定 Ⅳ度骨髓抑制 4 男 61 non-GCB双表达 2 2 exon 8 1 DAC、R-DICE 部分缓解 无 5 女 63 non-GCB 4 2 exon 8 2 DAC、R-Gemox 部分缓解 Ⅳ度骨髓抑制
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