摘要
目的探究金松双黄酮联合CK2抑制剂CX-4945对脑胶质母细胞瘤U87细胞增殖与凋亡的影响及作用机制。方法体外培养脑胶质母细胞瘤U87细胞,分别用0.01、0.10、1.00、10.00、100.00 μmol/L浓度的金松双黄酮处理U87细胞;用1.25、2.50、5.00、10.00、20.00 μmol/L浓度的CX-4945处理U87细胞。将U87细胞分为对照组(不做任何处理)、金松双黄酮组(100.00 μmol/L金松双黄酮)、CX-4945组(5.00 μmol/L CX-4945)、金松双黄酮联合CX-4945组(100.00 μmol/L金松双黄酮+5.00 μmol/L CX-4945)。采用MTT法检测细胞存活率;Caspase3/7活力检测法和Annexin Ⅴ/ PI双染法检测细胞凋亡情况;蛋白质印迹法检测100.00 μmol/L金松双黄酮处理U87细胞后Notch1通路相关蛋白ICN1、HES1和DLL3表达情况。结果 0、0.01、0.10、1.00、10.00、100.00 μmol/L浓度的金松双黄酮处理U87细胞存活率分别为(100.00±6.30)%、(112.02±7.63)%、(140.84±6.73)%、(113.92±7.92)%、(102.60±7.12)%、(73.16±2.74)%,差异具有统计学意义(F=55.21,P<0.001);0 μmol/L与0.01、0.10、1.00、100.00 μmol/L浓度时的细胞存活率差异均具有统计学意义(P=0.009;P<0.001;P=0.003;P<0.001)。100.00 μmol/L浓度时的金松双黄酮可抑制U87细胞活性,而其他浓度则表现为U87细胞活性的增强或无明显作用。0、1.25、2.50、5.00、10.00、20.00 μmol/L浓度的CX-4945处理U87细胞存活率分别为(100.00±5.53)%、(108.70±10.24)%、(93.14±2.82)%、(81.46±4.92)%、(56.92±3.99)%、(31.24±2.67)%,差异具有统计学意义(F=135.18,P<0.001);0 μmol/L与1.25、5.00、10.00、20.00 μmol/L浓度时的差异均具有统计学意义(P=0.022;P<0.001;P<0.001;P<0.001)。低浓度(1.25 μmol/L)的CX-4945对U87细胞活性表现为增强,较高浓度(5.00、10.00、20.00 μmol/L)时表现为抑制。对照组、金松双黄酮组、CX-4945组及金松双黄酮联合CX-4945组U87细胞的存活率分别为(100.00±5.53)%、(71.96±2.10)%、(77.66±4.12)%、(42.56±4.22)%,差异具有统计学意义(F=160.56,P<0.001);对照组与各处理组间的差异均具有统计学意义(均P<0.001);金松双黄酮联合CX4945处理组与金松双黄酮组、CX-4945组差异均具有统计学意义(均P<0.001)。上述4组U87细胞的Caspase3/7活力分别为2.34±0.47、4.02±0.22、3.67±0.32、5.85±0.28,差异具有统计学意义(F=55.80,P<0.001);各组的细胞凋亡率分别为(0.40±0.10)%、(17.37±0.57)%、(3.00±0.66)%、(33.47±0.87)%,差异具有统计学意义(F=1 822.18,P<0.001);进一步两两比较发现,对照组与各处理组的Caspase3/7活力、细胞凋亡率差异均具有统计学意义(P<0.001,P=0.001,P<0.001;P<0.001,P=0.001,P<0.001);金松双黄酮联合CX4945组与金松双黄酮组、CX-4945组的Caspase3/7活力、细胞凋亡率差异均具有统计学意义(均P<0.001)。100.00 μmol/L金松双黄酮处理U87细胞后Notch1通路相关蛋白ICN1(0.55±0.07vs.1.01±0.09)、HES1(0.66±0.08vs.1.00±0.06)和DLL3(0.74±0.04vs.1.01±0.09)的蛋白相对表达量均降低,差异均具有统计学意义(t=5.94,P=0.004;t=5.15,P=0.007;t=4.00,P=0.016)。结论金松双黄酮可通过抑制Notch1信号通路,与CK2抑制剂CX-4945协同抑制胶质母细胞瘤U87细胞增殖,促进细胞凋亡。
Objective To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods Glioblastoma U87 cells were cultured in vitro,and treated with 0.01,0.10,1.00,10.00,100.00μmol/L of sciadopitysin respectively.U87 cells were treated with 1.25,2.50,5.00,10.00,20.00μmol/L of CX-4945.U87 cells were divided into control group(without any treatment),sciadopitysin group(100.00μmol/L of sciadopitysin),CX-4945 group(5.00μmol/L of CX-4945),sciadopitysin combined with CX-4945 group(100.00μmol/L of sciadopitysin plus 5.00μmol/L of CX-4945).MTT method was used to detect cell viability,Caspase3/7 activity assay and AnnexinⅤ/PI double staining were used to detect cell apoptosis,and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1,HES1 and DLL3.Results The cell viabilities of U87 cells treated with 0,0.01,0.10,1.00,10.00,100.00μmol/L of sciadopitysin were(100.00±6.30)%,(112.02±7.63)%,(140.84±6.73)%,(113.92±7.92)%,(102.60±7.12)%and(73.16±2.74)%respectively,and there was a statistically significant difference(F=55.21,P<0.001).There were statistically significant differences in the cell viabilities of U87 cells between 0μmol/L and 0.01,0.10,1.00,100.00μmol/L of sciadopitysin treatment(P=0.009;P<0.001;P=0.003;P<0.001).The cell viability of U87 cells was inhibited by 100.00μmol/L of sciadopitysin,while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect.The cell viabilities of U87 cells treated with 0,1.25,2.50,5.00,10.00,20.00μmol/L of CX-4945 were(100.00±5.53)%,(108.70±10.24)%,(93.14±2.82)%,(81.46±4.92)%,(56.92±3.99)%and(31.24±2.67)%respectively,and there was a statistically significant difference(F=135.18,P<0.001).There were statistically significant differences in the cell viabilities of U87 cells between 0μmol/L and 1.25,5.00,10.00,20.00μmol/L of CX-4945 treatment(P=0.022;P<0.001;P<0.001;P<0.001).Low concentration(1.25μmol/L)of CX-4945 enhanced the cell viability of U87 cells,however higher concentrations(5.00,10.00,20.00μmol/L)of CX-4945 shown inhibitory effect.The cell viabilities of U87 cells in the control group,sciadopitysin group,CX-4945 group and sciadopitysin combined with CX-4945 group were(100.00±5.53)%,(71.96±2.10)%,(77.66±4.12)%and(42.56±4.22)%respectively,and there was a statistically significant difference(F=160.56,P<0.001).There were statistically significant differences between the control group and each treatment groups(all P<0.001).There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group,CX-4945 group(both P<0.001).The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47,4.02±0.22,3.67±0.32 and 5.85±0.28 respectively,and there was a statistically significant difference(F=55.80,P<0.001).The apoptosis rates of each groups were(0.40±0.10)%,(17.37±0.57)%,(3.00±0.66)%and(33.47±0.87)%respectively,and there was a statistically significant difference(F=1822.18,P<0.001).Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups(P<0.001,P=0.001,P<0.001;P<0.001,P=0.001,P<0.001).There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group,CX-4945 group(all P<0.001).The protein expression levels of Notch 1 pathway related proteins ICN1(0.55±0.07 vs.1.01±0.09),HES1(0.66±0.08 vs.1.00±0.06)and DLL3(0.74±0.04 vs.1.01±0.09)in U87 cells decreased significantly after treatment with 100.00μmol/L of sciadopitysin(t=5.94,P=0.004;t=5.15,P=0.007;t=4.00,P=0.016).Conclusion Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.
作者
连海伟
杨烁锐
刘仁忠
Lian Haiwei;Yang Shuorui;Liu Renzhong(Department of Neurosurgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《国际肿瘤学杂志》
CAS
2022年第6期321-326,共6页
Journal of International Oncology
基金
中央高校基本科研业务费专项资金(2042018kf0080)。
