Abstract:

Objective To discuss the effect of urolithin C （UC） on the proliferation， apoptosis， and autophagy of the acute myeloid leukemia （AML） HL-60 cells， and to clarify its mechanism. Methods The HL-60 cells were divided into different concentrations （20， 40， 60， 80， and 100 μmol·L^－1） of urolithin A （UA） groups， urolithin B （UB） groups， and UC groups. CCK-8 assay was used to detect the proliferation activity of the cells in various groups； the morphology of the cells in different concentrations of UC groups was observed under optical microscope. The HL-60 cells were divided into different concentrations （0， 20， 40， and 80 μmol·L^－1） of UC groups and 3-methyladenine （3-MA） combined with different concentrations （0， 20， 40， and 80 μmol·L^－1） of UC groups. CCK-8 assay was used to detect the proliferation activities of the cells in various groups.The HL-60 cells were divided into control group （0 μmol·L^－1） and different concentrations （20， 40， and 80 μmol·L^－1） of UC groups. The live/dead cell staining method was used to detect the dead rates of the cells in various groups； flow cytometry was used to detect the apoptotic rates of the cells in various groups；the autophagy of the cells was detected by autophagy staining kit （monodansylcadaverine， MDC）method； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of Beclin 1， autophagy related gene 9 （ATG9）， and autophagy related gene 7 （ATG7） mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of cysteinyl aspartate specific proteinase-3 （Caspase-3）， cleaved cysteinyl aspartate specific proteinase-3 （Cleaved Caspase-3）， microtubule-associated protein 1 light 3 （LC-3）， extracellular regulated protein kinases （ERK）， phosphorylated ERK （p-ERK）， AMP-activated protein kinase （AMPK）， and phosphorylated AMPK （p-AMPK） in the cells in various groups. Results The CCK-8 assay results showed that after cultured for 24， 48， and 72 h， compared with 0 μmol·L^－1 UA， UB， and UC groups， the proliferation activities of the cells in different concentrations of UA， UB， and UC groups were decreased （P<0.01） with a concentration-and time-dependent manner； at 48 h， compared with UA and UB， the half-maximal inhibitory concentration （IC₅₀） of UC was the lowest.The cell morphology observation results showed that compared with control group， the intercellular connection and the number of the cells were decreased with the increasing of UC concentration， and the cell fragment was increased. The CCK-8 assay results showed that compared with 40 and 80 μmol·L^－1 UC groups，the proliferation activities of the cells in 3-MA combined with 40 and 80 μmol·L^－1 UC groups were increased （P<0.05 or P<0.01）. The live/dead cell staining results showed that compared with control group， the dead rates of the cells in 40 and 80 μmol·L^－1 UC groups were increased （P<0.01）. The flow cytometry results showed that compared with control group， the apoptotic rate of the cells in 80 μmol·L^－1 UC group was increased （P<0.01）. The MDC method results showed that with the increasing of UC concentration， the green fluorescence in the cells in different concentrations of UC groups was gradually intensified. The RT-qPCR results showed that compared with control group， the expression levels of Beclin 1， ATG9， and ATG7 mRNA in the cells in 80 μmol·L^－1 UC group were increased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of Cleaved Caspase-3 protein in the cells in 20， 40， and 80 μmol·L^－1 UC groups were increased （P<0.01）， the ratio of membrane LC3 / cytoplasmic LC3 （LC3-Ⅱ/LC3-Ⅰ） in the cells in 80 μmol·L^－1 UC group was increased （P<0.05）， and the ratios of p-AMPK/AMPK and p-ERK/ERK in the cells in 40 and 80 μmol·L^－1 UC groups were increased （P<0.01）. Conclusion UC can inhibit the proliferation of the AML HL-60 cells，induce the apoptosis and autophagy， and increase the phosphorylation levels of ERK and AMPK proteins in the cells.

Key words: Urolithin C, Leukemia cell, Cell proliferation, Apoptosis, Cell autophagy