Abstract:

Objective To investigate the effects of Juglone on the apoptosis of osteosarcoma（OS） cells （U2OS and MG63 cells） through the cysteinyl aspartate specific proteinase-3（Caspase-3）/gasdermin E（GSDME）-mediated pyroptosis pathway. Methods The U2OS and MG63 cells were cultured in vitro and divided into control group， different concentrations （5， 10 and 20 μmol·L^-1） of Juglone groups and Caspase-3 inhibitor Z-DEVD-FMK group （10 μmol·L^-1 Juglone + 30 μmol·L^-1 Z-DEVD-FMK）. The survival rates of cells in various groups were assessed by cell counting kit-8（CCK-8） assay， and the apoptotic rates were detected by flow cytometry. Lactate dehydrogenase （LDH） release assay was used to measure the release rates of LDH from the cells. Western blotting method was used to detect the expression levels of apoptosis-related proteins including B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein（Bax）， cleaved-Caspase-3 and poly（ADP-ribose） polymerase（PARP）and pyroptosis-related proteins including GSDME full form（GSDME-F） and GSDME N-terminal（GSDME-N）. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in the cell supernataut in various groups were measured by enzyme-linked immunosorbent assay（ELISA） method. Results Compared with control group， the survival rates of cells in 5， 10， and 20 μmol·L^-1 Juglone groups were significantly decreased （P<0.05 or P<0.01）， and the 50% inhibitory concentration （IC₅₀） values of U2OS cells and MG63 cells were 8.4 and 10.2 μmol·L^-1， respectively. Compared with control group，the apoptotic rates and LDH release rates of U2OS and MG63 cells in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.05 or P<0.01）. Compared with control group， the expression levels of Bax， cleaved-Caspase-3， and cleaved-PARP proteins in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.01）， while the expression levels of Bcl-2 protein were significantly decreased （P<0.01）. Compared with control group， the levels of IL-1β and IL-18 in cell supernatant in 5 and 10 μmol·L^-1 Juglone groups were increased（P<0.01）. Compared with control group， the expression levels of cleaved-Caspase-3 and GSDME-N proteins in 5 and 10 μmol·L^-1 Juglone groups were significantly increased （P<0.01）， while there was no difference in the expression level of GSDME-F protein （P>0.05）. Compared with 10 μmol·L^-1 Juglone group， the expression levels of cleaved-Caspase-3 and GSDME-N in Z-DEVD-FMK group were significantly decreased（P<0.01），while there was no difference in the expression level of GSDME-F protein （P>0.05）. Conclusion Juglone can induce the apoptosis of U2OS and MG63 cells and cause the Caspase-3/GSDME-mediated pyroptosis.

Key words: Juglone, Osteosarcoma, Apoptosis, Cell pyroptosis, B-cell lymphoma-2, B-cell lymphoma-2-associated X protein