马钱子碱磷酸化c-Jun诱导人多发性骨髓瘤U266细胞凋亡研究
摘要
目的
多发性骨髓瘤(Multiple Myeloma,MM)是浆细胞恶性克隆大量增生,合成和分泌结构均一的单克隆免疫球蛋白和(或)其轻链的恶性肿瘤。尽管大剂量化疗、自体干细胞移植等新方法使缓解率和生存期有所改善,但由于瘤细胞增殖比例低(通常<1.5)﹪及瘤细胞多药耐药,MM的治疗反应较差,最终会出现耐药而复发。马钱子有效成分为生物碱类,马钱子碱是其主要成分之一,具有抗肿瘤作用,可引起HepG2细胞凋亡。本实验将马钱子碱作用于人多发性骨髓瘤U266细胞株,观察其对U266细胞株的生长影响及初步探讨抑制多发性骨髓瘤U266细胞株生长的分子机制。
方法
取对数生长期的U266细胞加入不同浓度(0 mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml)的马钱子碱作用24、48、72小时后用MTT法检测其的生长抑制率并计算48小时的IC_(50) ,同时观察细胞形态。利用流式细胞术观察马钱子碱作用后的细胞周期及线粒体膜电位的变化判断其细胞死亡的方式。利用半定量RT-PCR方法分别检测Bcl-2、Bax、caspase-3、c-Jun、cyt-c mRNA水平表达量的变化。
结果
1马钱子碱可抑制人多发性骨髓瘤U266细胞株生长,其抑制作用成时间和浓度依赖性(P<0.05),其48小时的IC_(50)为0.16mg/ml。
2利用RT-PCR检测马钱子碱作用U266细胞株中促凋亡基因Bax、抗凋亡基因Bcl-2的表达,结果显示Bax随时间表达量增加、Bcl-2时间表达量减少(P<0.05)。
3在荧光显微镜下可看到典型的细胞凋亡小体,经流式细胞仪检测不同浓度作用24小时后的U266细胞,可见到典型的Sub-G0/G1细胞群,其凋亡率随浓度增加而增加(P<0.05)。
4利用流式细胞仪检测不同浓度马钱子碱作用后的线粒体膜电位无明显下降(P>0.05)。
5加入caspase-8及caspase-9特异性抑制剂z-IETD-fmk、z-LEHD-fmk利用RT-PCR检测caspase-3、cyto-c基因表达量的变化,结果显示加入z-IETD-fmk后caspase-3表达量明显下降,而加入z-LEHD-fmk组caspase-3基因表达量无明显下降。
6分别加入马钱子碱、马钱子碱与JNK特异性抑制剂SP600125经RT-PCR检测caspase-3及c-Jun基因表达变化,结果显示JNK特异性抑制剂能明显阻断马钱子碱对caspase-3及c-Jun的激活,使其表达量明显下降。
结论
1马钱子碱对人多发性骨髓瘤U266细胞具有抑制增殖作用。
2马钱子碱通过诱导凋亡发挥其抑制U266细胞增殖的作用。
3马钱子碱可激活促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达。
4马钱子碱经死亡受体途径激活caspase-8进而激活凋亡执行者caspase-3诱导U266细胞凋亡。
5马钱子碱通过JNK信号通路磷酸化c-Jun诱导U266细胞凋亡。
Objective:
Multiple myeloma is a malignant clone of plasma cells proliferated, synthesis and secretion of monoclonal immunoglobulin uniform structure and (or) the light chain of cancer. Active ingredient of strychnos alkaloids, brucine is one of its main components, has anti-tumor effect, can cause apoptosis in HepG2 cells, which may be related to mitochondrial depolarization. In this study, the role of brucine on human multiple myeloma U266 cells lines were observed on the growth and to explore its molecular mechanism of suppressing the multiple myeloma U266 cells line growth.
Methods:
Logarithmic growth phase of the U266 cells with different concentrations(0 mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml),the effect of brucine 24,48,72 hours was detected by MTT growth inhibition rate and calculate the IC50 of 48 hours. Cell morphology was also observed. Observing cell cycle and mitochondrial membrane potential by flow cytometry determine the manner of cell death after the role of brucine. Detecting Bcl-2、Bax、caspase-3、c-Jun、cycto-c expression changes.
Results:
1 The apoptotic effect of brucine show a dose and time dependent manner (p<0.05). 48 hour IC50 0.16mg/ml.
2 Using RT-PCR, the role of brucine in the U266 cell line apoptosis gene Bax, Bcl-2 anti-apoptotic gene expression, the results show the expression of Bax increased with time, Bcl-2 expression decreased the time(P<0.05).
3 Typical apoptotic bodies can be seen under the fluorescence microscope. By flow cytometry with different concentrations for 24 hours after the U266 cells, can see the typical sub-G0/G1 cell groups. The apoptosis rate increased with the concentration (P<0.05). So brucine play a role in the inhibition of U266 cells by inducing apoptosis.
4 Mitochondrial membrane potential after different concentration of brucine had no significant decrease using flow cytometry.
5 At the same time, detecting caspase-3、cyto-c gene expression changes combined with caspase-8 and caspase-9 inhibitor z-IETD-fmk、z-LETD-fmk show joins z-IETD-fmk, the caspase-3 expression quantity obvious drop, but joins z-LETD-fmk the caspase-3 expression gene expression quantity to drop not obviously.
6 Detecting caspse-3 and c-Jun gene expression respectively after joining brucine、brucine and JNK specific inhibitor SP600125 by RT-PCR show that brucine induce apoptosis in U266 cells by JNK signaling pathway with activating caspase-3. The result showed that JNK inhibitor significantly blocked by brucine on the caspase-3 and c-Jun actibvation their expression decreased significantly.
Conclusion:
1 Brucine inhibits proliferation of human multiple myeloma cells line U266.
2 Brucine by inducing apoptosis to play its role in inhibiting the proliferation of U266 cells.
3 Brucine can activate the expression of pro-apoptotic gene Bax, inhibition of anti-apoptotic Bcl-2 gene expression.
4 Brucine by the death receptor pathway in turn activates caspase-8 activation of the apoptosis executor caspase-3 induced apoptosis in U266 cells.
5 Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells.
多发性骨髓瘤(Multiple Myeloma,MM)是浆细胞恶性克隆大量增生,合成和分泌结构均一的单克隆免疫球蛋白和(或)其轻链的恶性肿瘤。尽管大剂量化疗、自体干细胞移植等新方法使缓解率和生存期有所改善,但由于瘤细胞增殖比例低(通常<1.5)﹪及瘤细胞多药耐药,MM的治疗反应较差,最终会出现耐药而复发。马钱子有效成分为生物碱类,马钱子碱是其主要成分之一,具有抗肿瘤作用,可引起HepG2细胞凋亡。本实验将马钱子碱作用于人多发性骨髓瘤U266细胞株,观察其对U266细胞株的生长影响及初步探讨抑制多发性骨髓瘤U266细胞株生长的分子机制。
方法
取对数生长期的U266细胞加入不同浓度(0 mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml)的马钱子碱作用24、48、72小时后用MTT法检测其的生长抑制率并计算48小时的IC_(50) ,同时观察细胞形态。利用流式细胞术观察马钱子碱作用后的细胞周期及线粒体膜电位的变化判断其细胞死亡的方式。利用半定量RT-PCR方法分别检测Bcl-2、Bax、caspase-3、c-Jun、cyt-c mRNA水平表达量的变化。
结果
1马钱子碱可抑制人多发性骨髓瘤U266细胞株生长,其抑制作用成时间和浓度依赖性(P<0.05),其48小时的IC_(50)为0.16mg/ml。
2利用RT-PCR检测马钱子碱作用U266细胞株中促凋亡基因Bax、抗凋亡基因Bcl-2的表达,结果显示Bax随时间表达量增加、Bcl-2时间表达量减少(P<0.05)。
3在荧光显微镜下可看到典型的细胞凋亡小体,经流式细胞仪检测不同浓度作用24小时后的U266细胞,可见到典型的Sub-G0/G1细胞群,其凋亡率随浓度增加而增加(P<0.05)。
4利用流式细胞仪检测不同浓度马钱子碱作用后的线粒体膜电位无明显下降(P>0.05)。
5加入caspase-8及caspase-9特异性抑制剂z-IETD-fmk、z-LEHD-fmk利用RT-PCR检测caspase-3、cyto-c基因表达量的变化,结果显示加入z-IETD-fmk后caspase-3表达量明显下降,而加入z-LEHD-fmk组caspase-3基因表达量无明显下降。
6分别加入马钱子碱、马钱子碱与JNK特异性抑制剂SP600125经RT-PCR检测caspase-3及c-Jun基因表达变化,结果显示JNK特异性抑制剂能明显阻断马钱子碱对caspase-3及c-Jun的激活,使其表达量明显下降。
结论
1马钱子碱对人多发性骨髓瘤U266细胞具有抑制增殖作用。
2马钱子碱通过诱导凋亡发挥其抑制U266细胞增殖的作用。
3马钱子碱可激活促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达。
4马钱子碱经死亡受体途径激活caspase-8进而激活凋亡执行者caspase-3诱导U266细胞凋亡。
5马钱子碱通过JNK信号通路磷酸化c-Jun诱导U266细胞凋亡。
Objective:
Multiple myeloma is a malignant clone of plasma cells proliferated, synthesis and secretion of monoclonal immunoglobulin uniform structure and (or) the light chain of cancer. Active ingredient of strychnos alkaloids, brucine is one of its main components, has anti-tumor effect, can cause apoptosis in HepG2 cells, which may be related to mitochondrial depolarization. In this study, the role of brucine on human multiple myeloma U266 cells lines were observed on the growth and to explore its molecular mechanism of suppressing the multiple myeloma U266 cells line growth.
Methods:
Logarithmic growth phase of the U266 cells with different concentrations(0 mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml),the effect of brucine 24,48,72 hours was detected by MTT growth inhibition rate and calculate the IC50 of 48 hours. Cell morphology was also observed. Observing cell cycle and mitochondrial membrane potential by flow cytometry determine the manner of cell death after the role of brucine. Detecting Bcl-2、Bax、caspase-3、c-Jun、cycto-c expression changes.
Results:
1 The apoptotic effect of brucine show a dose and time dependent manner (p<0.05). 48 hour IC50 0.16mg/ml.
2 Using RT-PCR, the role of brucine in the U266 cell line apoptosis gene Bax, Bcl-2 anti-apoptotic gene expression, the results show the expression of Bax increased with time, Bcl-2 expression decreased the time(P<0.05).
3 Typical apoptotic bodies can be seen under the fluorescence microscope. By flow cytometry with different concentrations for 24 hours after the U266 cells, can see the typical sub-G0/G1 cell groups. The apoptosis rate increased with the concentration (P<0.05). So brucine play a role in the inhibition of U266 cells by inducing apoptosis.
4 Mitochondrial membrane potential after different concentration of brucine had no significant decrease using flow cytometry.
5 At the same time, detecting caspase-3、cyto-c gene expression changes combined with caspase-8 and caspase-9 inhibitor z-IETD-fmk、z-LETD-fmk show joins z-IETD-fmk, the caspase-3 expression quantity obvious drop, but joins z-LETD-fmk the caspase-3 expression gene expression quantity to drop not obviously.
6 Detecting caspse-3 and c-Jun gene expression respectively after joining brucine、brucine and JNK specific inhibitor SP600125 by RT-PCR show that brucine induce apoptosis in U266 cells by JNK signaling pathway with activating caspase-3. The result showed that JNK inhibitor significantly blocked by brucine on the caspase-3 and c-Jun actibvation their expression decreased significantly.
Conclusion:
1 Brucine inhibits proliferation of human multiple myeloma cells line U266.
2 Brucine by inducing apoptosis to play its role in inhibiting the proliferation of U266 cells.
3 Brucine can activate the expression of pro-apoptotic gene Bax, inhibition of anti-apoptotic Bcl-2 gene expression.
4 Brucine by the death receptor pathway in turn activates caspase-8 activation of the apoptosis executor caspase-3 induced apoptosis in U266 cells.
5 Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells.
引文
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