阿帕替尼联合阿霉素对外周T细胞淋巴瘤的抑制作用及机制研究

目的探讨抗血管生成药物阿帕替尼与常用化疗药物阿霉素联合应用对外周T细胞淋巴瘤Hut78细胞株的抑制作用以及相关分子作用机制,为开发外周T细胞淋巴瘤治疗新方法提供实验依据。方法将Hut78细胞分为不同浓度药物处理组:阿帕替尼单药(10、20、40、60μmol·L^-1)、阿霉素单药(1、2、4μmol·L^-1)、阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1、(60+1)μmol·L^-1、(60+2)μmol·L^-1],分别作用72 h,采用CCK-8法检测药物对细胞的增殖抑制作用。采用流式细胞术检测不同浓度药物处理组{阿帕替尼单药(40μmol·L^-1)、阿霉素单药(1、2μmol·L^-1)、阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1]}作用24 h后对Hut78细胞凋亡的影响。采用流式细胞术检测不同浓度阿帕替尼(10、20、40μmol·L^-1)作用24 h后对Hut78细胞周期的影响。采用蛋白印迹法检测不同浓度药物处理组{阿帕替尼单药(40μmol·L^-1)、阿霉素单药(1、2μmol·L^-1)、阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1]}作用24 h后,Hut78细胞中PI3K/Akt信号转导通路相关蛋白PI3K、p-PI3K、Akt、p-Akt及凋亡相关蛋白Bcl-2、Casepase-3、Bax的表达变化。结果不同浓度(10、20、40、60μmol·L^-1)的阿帕替尼作用72 h后,细胞抑制率分别为(13.42±2.19)%、(19.52±4.16)%、(31.49±3.16)%、(52.88±3.37)%,72 h的IC₅₀值为(59.34±0.31)μmol·L^-1,各药物处理组与对照组两两比较,差异具有统计学意义(χ²=10.116,P=0.018);不同浓度(1、2、4μmol·L^-1)的阿霉素分别作用72 h后,细胞抑制率分别为(15.82±3.23)%、(31.70±4.79)%、(42.34±5.23)%,72 h的IC₅₀值为(5.52±0.18)μmol·L^-1,各药物处理组与对照组两两比较,差异具有统计学意义(χ²=10.532,P=0.015);阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1、(60+1)μmol·L^-1、(60+2)μmol·L^-1]作用72 h的细胞抑制率分别为(51.70±2.09)%、(56.62±4.83)%、(61.35±1.79)%、(65.13±3.88)%。两药的联合指数(CI)<1,说明二者具有药物协同作用。阿帕替尼单药(40μmol·L^-1)、阿霉素单药(1、2μmol·L^-1)作用24 h的细胞凋亡率分别为(15.65±0.75)%、(13.85±2.15)%、(23.60±1.30)%,阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1]作用24h的细胞凋亡率分别为(27.00±1.90)%、(33.20±2.30)%,各药物处理组与对照组(6.10±0.90)%]比较,差异有统计学意义(χ²=16.251,P=0.006)。不同浓度(10、20、40μmol·L^-1)的阿帕替尼作用24 h后,G₀/G₁期细胞比例随浓度升高而升高(49.27±0.45)%、(50.34±1.24)%、(59.16±1.23)%],S期细胞比例随浓度升高而降低(42.81±2.22)%、(39.19±2.71)%、(34.08±1.01)%],各药物处理组与空白对照组G₀/G₁期(39.26±0.65)%,S期(49.40±2.52)%]比较,差异具有统计学意义(P<0.05)。阿帕替尼单药(40μmol·L^-1)、阿霉素单药(1、2μmol·L^-1)以及阿帕替尼+阿霉素(40+1)μmol·L^-1、(40+2)μmol·L^-1]作用24 h后,PI3K/Akt信号通路相关蛋白PI3K、p-PI3K、p-Akt和抗凋亡蛋白Bcl-2的表达水平呈下降趋势,促凋亡蛋白Casepase-3和Bax的表达水平呈上升趋势,Akt蛋白的表达水平无明显变化,各药物处理组p-PI3K/PI3K、p-Akt/Akt、Bcl-2、Casepase-3及Bax蛋白表达水平与对照组比较,差异均有统计学意义(P<0.05)。结论阿帕替尼可抑制外周T细胞淋巴瘤细胞的增殖并诱导其凋亡,同时具有细胞周期阻滞作用,其作用可能是通过抑制PI3K/Akt信号通路激活实现的。阿帕替尼联合阿霉素对外周T细胞淋巴瘤细胞具有协同抑制作用。

Inhibitory effects of apatinib combined with adriamycin on peripheral T-cell lymphoma cells and its mechanism

Objective To explore the inhibitory effects of the anti-angiogenic drug apatinib and the commonly used chemotherapy drug adriamycin on the peripheral T-cell lymphoma (PTCL) cell line Hut78 and the related molecular mechanisms,in order to provide relevant experimental basis for the development of new treatment methods for PTCL.Methods Hut78 cells were divided into several groups with drug treatment of different concentrations:apatinib single agent (10,20,40,60μmol·L^-1);adriamycin single agent (1,2,4μmol·L^-1);apatinib+adriamycin(40+1)μmol·L^-1,(40+2)μmol·L^-1,(60+1)μmol·L^-1,(60+2)μmol·L^-1].After treatment for 72 h,CCK-8 method was used to detect the inhibitory effects of drugs on the cell proliferation.Flow cytometry was used to detect the effects of drugs on the apoptosis of Hut78 cells in different concentrations treatment groups{apatinib single agent (40μmol·L^-1),adriamycin single agent (1,2μmol·L^-1),apatinib+adriamycin(40+1)μmol·L^-1,(40+2)μmol·L^-1]}after24 h,as well as the effects on the cell cycle of Hut78 cells in groups with different concentrations of apatinib (10,20,40μmol·L^-1)after 24 h.Western blotting was used to detect the expressions of PI3K/Akt signal pathway-related proteins (PI3K,p-PI3K,Akt and p-Akt) and apoptosis-related proteins (Bcl-2,Casepase-3 and Bax) in Hut78 cells in groups with different concentrations of drugs{apatinib single agent (40μmol·L^-1),adriamycin single agent (1,2μmol·L^-1),apatinib+adriamycin(40+1)μmol·L^-1,(40+2)μmol·L^-1]}after 24 h.Results After treated with different concentrations (10,20,40,60μmol·L^-1) of apatinib for 72 h,the inhibition rates of Hut78 cells were (13.42±2.19)%,(19.52±4.16)%,(31.49±3.16)%,(52.88±3.37)%,respectively.The IC₅₀value at 72 h was (59.34±0.31)μmol·L^-1,and the differences were statistically significant between each treatment group and the control group (χ²=10.116,P=0.018).After treated with different concentrations (1,2,4μmol·L^-1) of adriamycin for 72 h,the inhibition rates of Hut78 cells were (15.82±3.23)%,(31.70±4.79)%,(42.34±5.23)%,respectively.And the IC₅₀value at 72 h was (5.52±0.18)μmol·L^-1,and the differences were statistically significant between each treatment group and the control group (χ²=10.532,P=0.015).After treated with apatinib+adriamycin(40+1)μmol·L^-1,(40+2)μmol·L^-1,(60+1)μmol·L^-1,(60+2)μmol·L^-1]for 72 h,the inhibition rates of Hut78 cells were (51.70±2.09)%,(56.62±4.83)%,(61.35±1.79)%,(65.13±3.88)%,respectively.The combination index (CI) value of the two drugs was<1,suggesting a synergistic effect of the two drugs.Moreover,the apoptosis rate of Hut78 cells in groups of apatinib single agent (40μmol·L^-1) and adriamycin single agent (1,2μmol·L^-1) after 24 h was respectively (15.65±0.75)%,(13.85±2.15)%,(23.60±1.30)%;while that was respectively (27.00±1.90)%,(33.20±2.30)%in the apatinib+adriamycin groups(40+1)μmol·L^-1,(40+2)μmol·L^-1].The differences between each treatment group and the control group(6.10±0.90)%]was statistically significant (χ²=16.251,P=0.006).In addition,after treated with different concentrations of apatinib(10,20,40μmol·L^-1) for 24 h,the ratio of G₀/G₁phase cells gradually increasedrespectively (49.27±0.45)%,(50.34±1.24)%,(59.16±1.23)%],while the proportion of S phase cells gradually decreasedrespectively (42.81±2.22)%,(39.19±2.71)%,(34.08±1.01)%].The differences were statistically significant between each drug treatment group and the blank control groupG₀/G₁phase ratio (39.26±0.65)%,S phase ratio (49.40±2.52)%](P<0.05).Besides,after treatment with apatinib single agent (40μmol·L^-1),adriamycin single agent (1,2μmol·L^-1),apatinib+adriamycin(40+1)μmol·L^-1,(40+2)μmol·L^-1]for 24 h,the expression levels of PI3K/Akt signaling pathway-related proteins (PI3K,p-PI3K,p-Akt) and anti-apoptotic protein (Bcl-2) showed a downward trend,while the expression levels of the apoptotic proteins (Casepase-3 and Bax) showed an upward trend,and the expression level of Akt protein did not show a significant change.The protein expression levels of p-PI3K/PI3K,p-Akt/Akt,Bcl-2,Casepase-3,and Bax in each drug treatment group were statistically different from those in the control group (P<0.05).Conclusion Apatinib can inhibit the proliferation of PTCL cells,induce their apoptosis,and have a cell cycle arrest effect.Apatinib may inhibit the activation of PI3K/Akt signaling pathway and play a role in killing PTCL cells.Apatinib combined with adriamycin can exert a synergistic inhibitory effect on peripheral T-cell lymphoma cells.