黄芪总苷诱导人白血病NB4细胞凋亡的机制

Objective To investigate the effect of total astragalosides (TA) on proliferation and apoptosis in human leukemia NB4 cells in vitro. Methods The NB4 cells were treated with TA at different concentrations for 48 h in culture. Growth inhibition rates were measured by CCK-8 method. Flow cytometry was used to explore the cell apoptosis and the activity of NF-κB and Akt during apoptosis. Results TA at different concentrations (200, 400, 600, 800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner ( P < 0. 05), and the inhibitory rates of TA on NB4 cells were (14. 54 ± 3. 20) % , (24.79 ±3.98)%, (57.28 ±4.71)% and (88.28 ±4.65)% , respectively. In terms of the induction of apoptosis, there was a significant difference between the TA group and blank control [(1.80±1.24)%, P<0.05]. At TA doses of 200, 400 and 600 mg/L, the apoptotic rates of NB4 cells were ( 10. 03 ± 3.31)% , (14.87 ±3.65)% , (23.45 ± 1.90) % , respectively. Besides, TA induced apoptosis of NB4 cells in a dose-dependent manner in the groups of 200 mg/L, 400 mg/L, 600 mg/L (P<0.05). But there was no significant difference in apoptotic rates between the groups of 800 mg/L and 600 mg/L [(23.45 ±1.90)%, P> 0.05]. In the group of 800 mg/L, the necrotic cells increased highly and the necrotic rate reached (45.65 ± 3.16)%. After TA treatment of NB4 cells at different concentrations (200, 400, 600 mg/L), the expression of NF-kB protein was significantly decreased compared with that of the blank control (9. 79 ±0. 95, P<0.05), while Akt protein was not significantly decreased (P>0.05). Conclusion TA can inhibit the growth of NB4 cells and induce apoptosis in NB4 cells through an Akt-independent NF-kB signaling pathway.     

黄芪总苷诱导人白血病NB4细胞凋亡的机制

目的 研究黄芪总苷(TA)对体外培养的人早幼粒细胞白血病细胞株NB4增殖及凋亡机制的影响.方法 将不同浓度的TA与NB4细胞共培养48 h,采用细胞增殖及细胞毒性检测试剂盒8(CCK-8)检测细胞生长抑制率,以流式细胞仪检测细胞凋亡率及凋亡过程中细胞内核因子κB(NF-κB)和蛋白激酶B(PKB,又称Akt)蛋白浓度的变化.结果 200、400、600、800 ms/L TA均可抑制NB4细胞生长,抑制率分别为(14.54±3.20)%、(24.79±3.98)%、(57.28±4.71)%和(88.28±4.65)%,呈浓度依赖性增加(P<0.05).200、400、600、800 mg/L TA组的细胞凋亡率分别为(10.03±3.31)%、(14.87±3.65)%、(23.45±1.90)%和(25.26 4-2.07)%,与对照组[(1.80±1.24)%]相比,差异均有统计学意义(P<0.05),且200、400、600 ms/L TA组的细胞凋亡率呈浓度依赖性增加(P<0.05);800 mg/L TA组的细胞凋亡率增加不明显,与600 ms/L TA组相比,差异无统计学意义(P>0.05),但出现了大量的坏死细胞,坏死率达(45.65±3.16)%.细胞凋亡过程中伴随NF-κB蛋白表达下降,不同浓度TA组与对照组差异均有统计学意义(P<0.05),而Akt蛋白的表达无明显变化(P>0.05).结论 黄芪总苷可以抑制NB4细胞增殖,并可以通过不依赖Akt的NF-κB信号通路来诱导NB4细胞凋亡.

Abstract：

Objective To investigate the effect of total astragalosides (TA) on proliferation and apoptosis in human leukemia NB4 cells in vitro. Methods The NB4 cells were treated with TA at different concentrations for 48 h in culture. Growth inhibition rates were measured by CCK-8 method. Flow cytometry was used to explore the cell apoptosis and the activity of NF-κB and Akt during apoptosis. Results TA at different concentrations (200, 400, 600, 800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner ( P < 0. 05), and the inhibitory rates of TA on NB4 cells were (14. 54 ± 3. 20) % , (24.79 ±3.98)%, (57.28 ±4.71)% and (88.28 ±4.65)% , respectively. In terms of the induction of apoptosis, there was a significant difference between the TA group and blank control [(1.80±1.24)%, P<0.05]. At TA doses of 200, 400 and 600 mg/L, the apoptotic rates of NB4 cells were ( 10. 03 ± 3.31)% , (14.87 ±3.65)% , (23.45 ± 1.90) % , respectively. Besides, TA induced apoptosis of NB4 cells in a dose-dependent manner in the groups of 200 mg/L, 400 mg/L, 600 mg/L (P<0.05). But there was no significant difference in apoptotic rates between the groups of 800 mg/L and 600 mg/L [(23.45 ±1.90)%, P> 0.05]. In the group of 800 mg/L, the necrotic cells increased highly and the necrotic rate reached (45.65 ± 3.16)%. After TA treatment of NB4 cells at different concentrations (200, 400, 600 mg/L), the expression of NF-kB protein was significantly decreased compared with that of the blank control (9. 79 ±0. 95, P<0.05), while Akt protein was not significantly decreased (P>0.05). Conclusion TA can inhibit the growth of NB4 cells and induce apoptosis in NB4 cells through an Akt-independent NF-kB signaling pathway.     

Effects of different time points of bortezomib on the pathway of NF-κB in drug-resistant K562 cells

Objective To study the effects of bortezomib on the expression of NF-κB, IκB and P-gp of drug-resistant K562 cells induced by daunorubicin (K562/DNR), to explore the molecular mechanism of drug-resistant reverse. Methods The expression of NF-κB, IκB and P-gp in K562/DNR cells were detected when the cells had been treated with 100 μg/ml DNR only or together with 4 μg/L bortezomib for 12 h, 24 h and 36 h. The apoptosis rates were detected in each group respectively and the activity of NF-κB was detected by ELISA method. Results Compared with the control group, the expressions of NF-κB and P-gp in K562/DNR could be increased and IκB was decreased after being treated with DNR. When K562/DNR were cultured with bortezomib, the expressions of NF-κB and P-gp induced by DNR were significantly suppressed and IκB was increased. The activity of NF-κB were detected in different time points: (15.3±1.87) %[(23.8± 2.27) % in DNR group] at 12 h, (10.2±1.69) % [(25.4±1.98) % in DNR group] at 24 h, (6.08±2.53) % [(26.9±2.58) % in DNR group] at 36 h. There were a significant differences between DNR group and DNR+PS-341group. The apoptosis rates were increased in DNR+PS-341 group at different time points than those in DNRgroup, (35.23±5.15) % [(15.56±4.12) % in DNR group] at 12 h, (40.26±6.89) % [(17.25±2.89) % in DNR group] at 24 h, (43.58±7.69) % [(22.47±4.58) % in DNR group] at 36 h. The effccts showed the character of time-dependent pattern. Conclusion Bortezomib could downregulate the expressions of NF-κB and P-gp in K562/DNR, reverse the drug resistance and up-regulate the apoptotic rates in K562/DNR cells.     

Effects of different time points of bortezomib on the pathway of NF-κB in drug-resistant K562 cells

Objective To study the effects of bortezomib on the expression of NF-κB, IκB and P-gp of drug-resistant K562 cells induced by daunorubicin (K562/DNR), to explore the molecular mechanism of drug-resistant reverse. Methods The expression of NF-κB, IκB and P-gp in K562/DNR cells were detected when the cells had been treated with 100 μg/ml DNR only or together with 4 μg/L bortezomib for 12 h, 24 h and 36 h. The apoptosis rates were detected in each group respectively and the activity of NF-κB was detected by ELISA method. Results Compared with the control group, the expressions of NF-κB and P-gp in K562/DNR could be increased and IκB was decreased after being treated with DNR. When K562/DNR were cultured with bortezomib, the expressions of NF-κB and P-gp induced by DNR were significantly suppressed and IκB was increased. The activity of NF-κB were detected in different time points: (15.3±1.87) %[(23.8± 2.27) % in DNR group] at 12 h, (10.2±1.69) % [(25.4±1.98) % in DNR group] at 24 h, (6.08±2.53) % [(26.9±2.58) % in DNR group] at 36 h. There were a significant differences between DNR group and DNR+PS-341group. The apoptosis rates were increased in DNR+PS-341 group at different time points than those in DNRgroup, (35.23±5.15) % [(15.56±4.12) % in DNR group] at 12 h, (40.26±6.89) % [(17.25±2.89) % in DNR group] at 24 h, (43.58±7.69) % [(22.47±4.58) % in DNR group] at 36 h. The effccts showed the character of time-dependent pattern. Conclusion Bortezomib could downregulate the expressions of NF-κB and P-gp in K562/DNR, reverse the drug resistance and up-regulate the apoptotic rates in K562/DNR cells.     

As4S4 Induced Apoptosis in HeLa Cells and Its Molecular Mechanism

Objective To investigate the As4S4 induced growth inhibition and apoptosis in HeLa cells and its possible relationship with cyclooxygenase-2 (COX-2). Methods HeLa cells were treated with various concentrations (7.5, 15, 30, 60 mg/L) of As4S4 at different times (12, 24, 36, 48, 60 h). Cell growth was measured by MTT. Apoptosis was detected by double staining flow cytometry (FCM). Levels of PGE2 were measured by radioimmunoassay. The expression of COX-2 protein was examined by Western blot analysis. Results After treated with different concentrations of As4S4, the growth of HeLa cells was suppressed significantly in a dose-and time-dependent manner. The IC50 of 24 h was 30 mg/L (P＜0.01). As4S4 induced apoptosis with apoptosis rates at 8.13%-62.36% by flow cytometry (FCM) in a dose-dependent manners. The release of PGE2 was reduced in HeLa cells with the values being (70.56±2.03), (48.58±2.28), (29.25±1.57) and (18.02±1.04) respectively, significantly different compared with control group (3.15±0.01) (P＜0.01). As4S4 also inhibited the activity and expression of COX-2 in a dose dependent manner and down-regulated the expression of COX-2 protein greatly. Conclusion As4S4 could inhibit the proliferation and increase apoptosis in human HeLa cells. These effects may depend on the inhibition of the expression of COX-2 and PGE2 by As4S4.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.