双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞活性、增殖、凋亡的影响及机制研究

国际肿瘤学杂志››2024,Vol. 51››Issue (3): 129-136.doi:10.3760/cma.j.cn371439-20231130-00021

双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞活性、增殖、凋亡的影响及机制研究

任露¹^,², 谢晓丽²^,³, 张坤⁴, 王丽娟²^,³^,⁵()

¹锦州医科大学临沂市人民医院培养基地，临沂 276000
²山东省临沂市人民医院中心实验室，临沂 276000
³山东省临沂市肿瘤生物学重点实验室，临沂 276000
⁴滨州医学院附属医院肿瘤科，滨州 256600
⁵山东省临沂市人民医院血液内科，临沂 276000

收稿日期:2023-11-30修回日期:2024-01-15出版日期:2024-03-08发布日期:2024-04-10
通讯作者:王丽娟，Email： wanglj730@163.com
基金资助:
山东省自然科学基金(ZR2022MH058);山东省自然科学基金(ZR2022-QH168)

Effects and mechanisms of dihydroartemisinin combined with carfilzomib on the activity， proliferation， and apoptosis of multiple myeloma cells

Ren Lu¹^,², Xie Xiaoli²^,³, Zhang Kun⁴, Wang Lijuan²^,³^,⁵()

¹Linyi People's Hospital Cultivation Base， Jinzhou Medical University， Linyi 276000， China
²Central Laboratory， Linyi People's Hospital of Shandong Province， Linyi 276000， China
³Linyi Key Laboratory of Tumor Biology of Shandong Province， Linyi 276000， China
⁴Department of Oncology， Binzhou Medical University Hospital， Binzhou 256600， China
⁵Department of Hematology， Linyi People's Hospital of Shandong Province， Linyi 276000， China

Received:2023-11-30Revised:2024-01-15Online:2024-03-08Published:2024-04-10
Contact:Wang Lijuan， Email： wanglj730@163.com
Supported by:
Natural Science Foundation of Shandong Province of China(ZR2022MH058);Natural Science Foundation of Shandong Province of China(ZR2022-QH168)

摘要/Abstract

摘要：

目的研究双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞株ARD活性、增殖和凋亡的影响及其可能机制。方法体外培养多发性骨髓瘤细胞ARD，分别用0、5、10、20、40、80 μg/ml浓度双氢青蒿素及0、5、10、20、40、80 nmol/L浓度卡非佐米处理ARD细胞。将ARD细胞分为对照组（不做任何处理）、双氢青蒿素组（2 μg/ml）、卡非佐米组（8 nmol/L）和联合组（双氢青蒿素2 μg/ml+卡非佐米8 nmol/L）。采用MTT法和EdU-488法检测细胞活性和增殖情况；活细胞/死细胞双染法和流式细胞术检测细胞凋亡情况；蛋白质印迹法检测细胞凋亡相关蛋白表达水平。结果经0、5、10、20、40、80 μg/ml浓度双氢青蒿素处理ARD细胞的存活率分别为（100.00±2.18）%、（50.22±3.09）%、（37.39±2.34）%、（30.42±1.79）%、（23.80±1.12）%、（18.04±0.79）%，差异有统计学意义（F=653.30，P<0.001）；随着药物浓度增加，ARD细胞活性逐渐下降（均P<0.05）。经0、5、10、20、40、80 nmol/L浓度卡非佐米处理ARD细胞的存活率分别为（100.00±1.12）%、（83.98±2.95）%、（67.27±2.10）%、（58.24±2.02）%、（46.34±1.14）%、（37.47±1.36）%，差异有统计学意义（F=227.40，P<0.001）；随着药物浓度增加，ARD细胞活性逐渐下降（均P<0.05）。对照组、双氢青蒿素组、卡非佐米组和联合组的细胞存活率分别为（100.00±2.67）%、（67.23±0.57）%、（76.23±2.83）%、（27.06±1.09）%，差异有统计学意义（F=655.60，P<0.001）；双氢青蒿素组、卡非佐米组、联合组与对照组相比，差异均有统计学意义（均P<0.001）；双氢青蒿素组、卡非佐米组与联合组相比，差异均有统计学意义（均P<0.001）。对照组、双氢青蒿素组、卡非佐米组和联合组细胞EdU阳性率分别为（100.00±8.17）%、（68.07±6.14）%、（85.04±2.78）%、（19.62±3.83）%，差异有统计学意义（F=115.20，P<0.001）；双氢青蒿素组、卡非佐米组、联合组与对照组相比，差异均有统计学意义（P<0.001；P=0.047；P<0.001）；双氢青蒿素组、卡非佐米组与联合组相比，差异均有统计学意义（均P<0.001）。活细胞/死细胞双染实验显示，在明场视野下，对照组细胞形态完整，各用药组细胞形态不规则、体积缩小、细胞质浓缩，细胞周围可见形态不规则的凋亡小体，其中，联合组变化最明显；在荧光视野下，对照组细胞只显示绿色荧光，各用药组均出现红色荧光，其中，联合组中红色荧光细胞占比最大。对照组、双氢青蒿素组、卡非佐米组和联合组的细胞凋亡率分别为（9.06±2.95）%、（29.50±1.34）%、（20.77±3.00）%、（58.23±5.13）%，差异有统计学意义（F=115.80，P<0.001）；双氢青蒿素组、卡非佐米组、联合组与对照组相比，差异均有统计学意义（P<0.001；P=0.012；P<0.001）；双氢青蒿素组、卡非佐米组与联合组相比，差异均有统计学意义（均P<0.001）。对照组、双氢青蒿素组、卡非佐米组和联合组的P53、Cleaved-Caspase-3、Bcl-2和Bax蛋白相对表达量差异均有统计学意义（F=21.76，P<0.001；F=42.87，P<0.001；F=44.27，P<0.001；F=163.50，P<0.001）；双氢青蒿素组、卡非佐米组、联合组与对照组相比，差异均有统计学意义（均P<0.05）；双氢青蒿素组、卡非佐米组与联合组相比，差异均有统计学意义（均P<0.05）。结论双氢青蒿素联合卡非佐米可协同抑制多发性骨髓瘤ARD细胞的活性及增殖能力，并促进其凋亡，其作用机制可能与线粒体凋亡途径相关。

关键词:多发性骨髓瘤,细胞增殖,细胞凋亡,双氢青蒿素,卡非佐米

Abstract:

ObjectiveTo study the effects and potential mechanisms of the combination of dihydroartemisinin and carfilzomib on the activity， proliferation， and apoptosis of multiple myeloma ARD cell lines.MethodsIn vitrocultivation of multiple myeloma ARD cells involved treating the cells with dihydroartemisinin at concentrations of 0， 5， 10， 20， 40， and 80 μg/ml， and with carfilzomib at concentrations of 0， 5， 10， 20， 40， and 80 nmol/L. The ARD cells were divided into a control group （no treatment）， a dihydroartemisinin group （2 μg/ml）， a carfizomib group （8 nmol/L）， and a combination group （dihydroartemisinin 2 μg/ml + carfizomib 8 nmol/L）. Cell activity and proliferation were assessed by MTT assay and EdU-488 assay； cell apoptosis was evaluated using live cell/dead cell dual staining and flow cytometry. The expression levels of apoptosis-related proteins were examined using Western blotting analysis.ResultsThe cell survival rates of ARD cells treated with 0， 5， 10， 20， 40， and 80 μg/ml dihydroartemisinin were （100.00±2.18）%，（50.22±3.09）%，（37.39±2.34）%，（30.42±1.79）%，（23.80±1.12）%， and （18.04±0.79）%， respectively， and there was a statistically significant difference （F=653.30，P<0.001）. With the increase of drug concentration， ARD cell activity decreased gradually （allP<0.05）. The cell survival rates of ARD cells treated with 0， 5， 10， 20， 40， and 80 nmol/L carfilzomib were （100.00±1.12）%，（83.98±2.95）%，（67.27±2.10）%，（58.24±2.02）%，（46.34±1.14）%， and （37.47±1.36）%， respectively， and there was a statistically significant difference （F=227.40，P<0.001）. With the increase of drug concentration， ARD cell activity decreased gradually （allP<0.05）. The cell survival rates for the control group， dihydroartemisinin group， carfilzomib group， and combination group were （100.00±2.67）%，（67.23±0.57）%，（76.23±2.83）%， and （27.06±1.09）%， respectively， and there was a statistically significant difference （F=655.60，P<0.001）. There were statistically significant differences in the dihydroartemisinin group， carfilzomib group， and combination group compared with control group （allP<0.001）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （bothP<0.001）. The EdU-488 experiment showed that the EdU-positive rates of ARD cells in the control group， dihydroartemisinin group， carfilzomib group， and combination group were （100.00±8.17）%，（68.07±6.14）%，（85.04±2.78）%， and （19.62±3.83）%， respectively， and there was a statistically significant difference （F=115.20，P<0.001）. There were statistically significant differences in the dihydroartemisinin group， carfilzomib group， and combination group compared with control group （P<0.001；P=0.047；P<0.001）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （bothP<0.001）. The live cell/dead cell dual staining experiment showed， under bright-field observation， the cell morphology was intact in the control group. In all the drug groups， the cell morphology became irregular， reduced in size with condensed cytoplasmic， and apoptotic vesicles with irregular morphology were seen around the cells， among which the most obvious changes were seen in the combination group. Under fluorescence observation， the cells in the control group only displayed green fluorescence. In all drug-treated groups， cells with red fluorescence were observed， with the combination group having the highest percentage of cells with red fluorescence among the total cell population. The apoptosis rates for the control group， dihydroartemisinin group， carfilzomib group， and combination group were （9.06±2.95）%，（29.50±1.34）%，（20.77±3.00）%， and （58.23±5.13）%， respectively， and there was a statistically significant difference （F=115.80，P<0.001）. There were statistically significant differences in the dihydroartemisinin group， carfilzomib group， and combination group compared with control group （P<0.001；P=0.012；P<0.001）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （bothP<0.001）. There were statistically significant differences in the relative expression levels of P53， Cleaved-Caspase-3， Bcl-2， and Bax proteins among the control group， dihydroartemisinin group， carfilzomib group， and combination group （F=21.76，P<0.001；F=42.87，P<0.001；F=44.27，P<0.001；F=163.50，P<0.001）. There were statistically significant differences in the dihydroartemisinin group， carfilzomib group， and combination group compared with control group （allP<0.05）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （bothP<0.05）.ConclusionThe combination of dihydroartemisinin and carfilzomib can synergistically inhibit the activity and proliferation of multiple myeloma ARD cells， and promote apoptosis， and the underlying mechanism may be associated with the mitochondrial apoptosis pathway.

Key words:Multiple myeloma,Cell proliferation,Apoptosis,Dihydroartemisinin,Carfilzomib

任露, 谢晓丽, 张坤, 王丽娟. 双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞活性、增殖、凋亡的影响及机制研究[J]. 国际肿瘤学杂志, 2024, 51(3): 129-136.

Ren Lu, Xie Xiaoli, Zhang Kun, Wang Lijuan. Effects and mechanisms of dihydroartemisinin combined with carfilzomib on the activity， proliferation， and apoptosis of multiple myeloma cells[J]. Journal of International Oncology, 2024, 51(3): 129-136.

图/表5

图1

图2

图3

组别	P53	Cleaved-Caspase-3	Bcl-2	Bax
对照组	1.00±0.17	1.00±0.27	1.00±0.09	1.00±0.27
双氢青蒿素组	2.54±0.72^ab	1.60±0.11^ab	0.65±0.12^ab	2.41±0.17^ab
卡非佐米组	2.66±0.58^ab	1.57±0.08^ab	0.67±0.05^ab	1.81±0.14^ab
联合组	4.40±0.41^a	2.83±0.27^a	0.20±0.08^a	4.47±0.20^a
F值	21.76	42.87	44.27	163.50
P值	<0.001	<0.001	<0.001	<0.001

表1

图4

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