塞来昔布抑制COX-2 和PD-1 发挥抗肝癌作用

目的:研究塞来昔布的抗肝癌作用及其潜在的分子机制。方法:选取2012 年2 月~2016 年6 月在我院进行手术治疗的原发性肝细胞癌(HCC)患者65 例作为研究对象,另选取肝胆外科提供的正常肝脏组织标本15 例作为对照。采用免疫组织化学法(IHC)检测肿瘤及正常组织样本中COX-2 与PD-1 的表达。采用Pearson 相关性分析HCC 患者COX-2 与PD-1 的相关性。建立H22 肝癌细胞BALB/ c 小鼠模型,随机分为对照组、塞来昔布组和PD-1 抗体组(每组15 只)。给药4 周后处死全部存活小鼠,取出肿瘤。绘制肿瘤生长曲线及小鼠生存曲线。采用IHC 分析各组肿瘤组织中COX-2、PD-1 的表达水平及CD8+ T 与Foxp3 阳性Treg 细胞数。流式细胞术检测外周血中CD8+ T 与CD4+ CD25+ Treg 细胞数量。分离正常BALB/ c 小鼠的外周血单核细胞(PBMC),分别转染PD-1 及对照siRNA,使用塞来昔布处理后,Western blot 检测COX-2、PD-1、CD8 及Foxp3水平。结果:IHC 结果显示HCC 患者肿瘤组织中COX-2 与PD-1 的表达水平均显著高于对照组(P<0.001)。Pearson 相关性分析显示HCC 患者肿瘤组织中COX-2 的表达水平与PD-1 呈显著正相关(R2 =0.673,P<0.001)。塞来昔布、PD-1 抗体治疗小鼠的肿瘤生长曲线和生存曲线均显著优于对照组(P<0.05),塞来昔布组、PD-1 抗体组小鼠的肿瘤生长曲线和生存曲线相比差异均无统计学意义(P>0.05)。IHC 与流式分析显示,塞来昔布能显著降低肿瘤组织中PD-1 的表达(P<0.05);塞来昔布与PD-1 抗体均能使肿瘤组织及外周血CD8+ T 细胞显著增多(P<0.05),使Treg 细胞显著减少(P<0.05)。Western blot 结果显示,塞来昔布能显著降低小鼠PBMC 的COX-2、PD-1、CD8 及Foxp3 水平,而不影响转染PD-1 siRNA 后PBMC 的CD8 及Foxp3水平。结论:HCC 患者肿瘤组织中COX-2 与PD-1 均显著增高,塞来昔布可能通过抑制COX-2 调节PD-1影响肿瘤免疫从而发挥抗肝癌的作用。

Antihepatocarcinoma effect of Celecoxib by inhibiting COX-2 and PD-1

Objective:To investigate the role of celecoxib in inhibiting liver cancer and to explore the potential molecular mechanisms.Methods:A total of 65 cases of patients with primary hepatocellular carcinoma(HCC) underwent surgical treatment in our hospital from February 2012 to June 2016 were recruited as study object.Another 15 cases of normal liver tissue offered by the department of hepatobiliary surgery were selected as control.Immunohistochemical staining(IHC) was used to detect the expression level of COX-2 and PD-1 in tumor and normal tissue samples.Pearson correlation analysis were used to evaluate the correlation between COX-2 and PD-1 in HCC patients.Construction of H22 hepatoma cells bearing BALB/ c mice model,randomly divided them into control group,celecoxib group and PD-1 antibody group(15 mice for each group).Sacrificed all mice alive at the end of the 4th weeks after the treatment and removed the tumors then.The tumor growth curves and survival curves were drawn to observe the anti-tumor effect.IHC were used to evaluate the expression of COX-2,PD-1 as well as the number of CD8+ T and foxp3 positive Treg cells in tumor tissue.Flow cytometry were used to determine the number of CD8+ T and CD4+ CD25+ Treg cells in peripheral blood.The peripheral blood mononuclear cell(PBMC) of BALB/ c mice were separated and scramble or PD-1 siRNA were transfected then,Western blot analysis were used to detected the level of COX-2,PD-1,CD8 and Foxp3 after the treatment of celecoxib.Results:IHC results showed that the expression level of COX-2 and PD-1 in tumor tissue of HCC patients were significant higher than control (P < 0.001).Pearson correlation analysis showed COX-2 were both positive correlated with PD-1 in the tumor tissue of HCC patients(R2 = 0.673,P <0.001). The tumor growth curves and survival curves in celecoxib or PD-1 antibody groups were significant better than in control group (P<0.05).There were no significant difference of tumor growth curves and survival curves between celecoxib group and PD-1 antibody group(P>0.05).IHC and flow cytometry analysis showed that celecoxib treatment significant decreased the expression level of PD-1(P<0.05).Both celecoxib and PD-1 antibody treatment significant increased the number of CD8+ T cells while decreased the number of Treg cells in the tissue or the peripheral blood(P<0.05).Western blot analysis showed that celecoxib significant decreased the level of COX-2,PD-1, CD8 and Foxp3 in PBMCs, while it did not affect the level of CD8 and Foxp3 in PD-1 siRNA transfected PBMCs.Conclusion:The expression level of COX-2 and PD-1 were increased in the tumor tissue of HCC patients.Celecoxib may inhibit liver cancer by regulating PD-1 mediated tumor immune via inhibiting COX-2.