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Mechanism study on regulation of glioblastoma cell proliferation and apoptosis by sciadopitysin combined with CX-4945 through Notch1 pathway
Lian Haiwei, Yang Shuorui, Liu Renzhong
Journal of International Oncology 2022, 49 (6): 321-326. DOI:10.3760/cma.j.cn371439-20220118-00061
ObjectiveTo investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.
MethodsGlioblastoma U87 cells were cultured
in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group （without any treatment）, sciadopitysin group （100.00 μmol/L of sciadopitysin）, CX-4945 group （5.00 μmol/L of CX-4945）, sciadopitysin combined with CX-4945 group （100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945）. MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ 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.
ResultsThe cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/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 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/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 100.00 μmol/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, 20.00 μmol/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 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment （
P=0.022;
P<0.001;
P<0.001;
P<0.001）. Low concentration （1.25 μmol/L） of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations （5.00, 10.00, 20.00 μmol/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=1 822.18,
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 100.00 μmol/L of sciadopitysin （
t=5.94,
P=0.004;
t=5.15,
P=0.007;
t=4.00,
P=0.016）.
ConclusionSciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.
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