Abstract:

Objective To 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. Methods In vitro cultivation 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. Results The 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 （all P<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 （all P<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 （all P<0.001）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （both P<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 （both P<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 （both P<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 （all P<0.05）. There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group （both P<0.05）. Conclusion The 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