Xanthohumol Induced Acute Cell Death In HL-60 Cells Through Reactive Oxygen Species Burst In Serum-free System

BACKGROUND: Plant natural products play an important role in anti-tumor field.In recent years,Xanthohumol(XN),the natural product extracted from hops has gained considerable interest due to its extensive biological activities.Among them,the free radicals related mechanisms are often got researched first.As a flavonoid polyphenol compound,the antioxidant activity of XN has been extensively studied and reported since 2000.However,since 2007,the pro-oxidant activity of XN has also been found in a variety of cells.Our previous study also found a potent reactive oxygen species(ROS)generation in HL-60 cells when treated with XN.However,why the antioxidant XN exhibited pro-oxidant activity in cells is still unclear.Therefore,it is very important to study the details of pro-oxidant activity of XN in cells.OBJECTIVE: To analyze the pro-oxidant activity of XN and its effect on cell fate in HL-60 cells.To explore the suitable cell model conditions for studying ROS generation mechanisms in cells.METHODS: In view that serum added in the normal cell culture medium exhibited a strong ROS scavenging activity;the serum-free system was chosen to detect ROS production.The ROS generation was detected with the fluorescence probe DCFH-DA by flow cytometry,and the dot plot data were used to analyze cell debris formation after treated with XN.Cell disruptions were further evaluated with the release of lactase dehydrogenase(LDH)assay,and the cell numbers were counted with Trypan Blue exclusion assay.The changes of cell morphology were observed by living cell imaging system,and membrane permeability was evaluated with PI fluorescence staining.Annexin V-FITC/PI staining assay was used to detect cell apoptosis.The release of IL-1?(a marker of pyroptosis)was detected by enzyme-linked immunosorbent assay(ELISA).The cleavage of caspase-3(marker of apoptosis)and GSDMD(marker of pyroptosis),and the phosphorylation of MLKL(marker of necroptosis)were checked with Western Blot analysis.RESULTS:(1)10 ?M XN could induce ROS burst in HL-60 cells,while the ROS level decreased sharply when treated with higher concentration of XN(20 and 40 ?M).Dot plot data analysis indicated that 20 and 40 ?M XN treatments resulted in a large number of fragments of HL-60 cells,suggesting a serious cell disruption.(2)The cell disruption was further confirmed with LDH release assay and trypan blue exclusion assay;LDH release increased and cell number decreased with XN treatments.The antioxidant N-acetyl-L-cysteine(NAC)intervention can reduce the release of LDH and the number of cell death to alleviate the situation.This indicated that the XN-induced cell disruption is related to the generation of ROS.(3)The results of living cell imaging showed that XN treatment induced the rapid swelling of HL-60 cells,and the PI permeability results showed the increase of cell membrane permeability;NAC intervention could effectively alleviate these effects.This indicated that ROS was related to this increase of cell membrane permeability.(4)In order to identify the type of this acute death induced by XN,further detected the occurrence of three main cell deaths: apoptosis,pyroptosis,and programmed necrosis.The results indicated that the acute cell death did not belong to any of these three major cell deaths.It is probably to be attributed to ROS outbreak caused acute cell necrosis.(5)Finally,we discussed the cell model for studying ROS generation mechanisms.It was found that 10% serum could completely mask XN-induced ROS in 10 ?M and 20 ?M,although cell debris disappeared simultaneously.The effect of different concentrations of serum(1% to 10%)on ROS generation under 10 ?M XN treatments were further evaluated,and the results showed that 2% of serum had already been able to completely mask the ROS generated.so it is suggested that serum is better to be avoided when studying the detailed mechanisms of ROS generation,such as the site of ROS generated.In view with the cell disruption caused in serum-free system,a proper concentration should be selected through evaluating the dose-dependent curve,and the ROS inflection point could be well-chosen as the optimal experimental concentration.For example,the ROS inflection point 10 ?M could be selected in our XN study.CONCLUSION:(1)XN induced ROS burst in HL-60 cells in serum-free system,and caused acute necrosis other than apoptosis,pyroptosis or necroptosis.(2)In view of the serum could cover the true level of ROS generation,the detailed mechanisms of ROS generation should be studied in serum-free system,and the inflection point of ROS formation should be selected as the optimal experimental concentration according to the dose-dependent curve.