| PSP(Polysaccharopeptide of Trametes versicolor)is an active component in Trametes versicolor.It has clinically applied as an immunomodulator for cancer patients.In recent years,it has been found that PSP can inhibit and directly kill many kinds of cancer cells by interfering their cell cycles and inducing apoptosis.Previous studies in our laboratory found that in addition to typical apoptotic morphological changes after PSP treatment,leukemic cells such as Molt-4 also showed cell swelling,suggesting that there might be other mechanisms during death.Therefore,this study intends to further explore and discuss this phenomenon.Microscope-micrometer was used to analyze the changes in diameter of Molt-4 cells induced by PSP.It was found that 20 ?g/mL PSP could increase the average diameter of Molt-4 cells from 10.80 ±1.40?m to 12.12 ± 2.06?m,while 40 ?g /mL PSP could increase to 13.01 ± 2.63 and 80?g/mL to 13.81 ± 2.52?m.Flow cytometric analysis of the forward scattering(FS)and side scattering(SS)signals showed the cell diameter had two different changes,including decrease and increase.Cells were incubated with PSP(20,40,80 ?g/ml)for 48 hours and labeled with FLICA,a caspase-1 fluorescent inhibitor then analyze the proportion of FLICA positive cells by Flow cytometry.Compare to 2.38% positive in the control group,it was found that FLICA positive populations increased to 4.57,5.25 and 16.40% respectively.At 12,24,36 and 48 h respectively,FLICA positive cells percentages had been increased from 3.2% in the control group to 8.65,12.4,18.6 and 22.8%,respectively(P<0.001).These results suggest that PSP can activate caspase-1 in molt-4 cells in a time and dose-dependent manner.Caspase-1 inhibitors VX-765 and AC-YVAD-CMK not only failed to inhibit caspase-1 activation and the cell death induced by PSP but also showed significant cytotoxicity in a range of 25 to 100 ?M.Flow cytometry was used to analyze the distribution of FLICA positive cells(FLICA+)on the FS/SS scatting diagram using a Gating technique.It was found that the percentage of cells with strong FS signal increased after PSP(80 ?g/mL)treatment at 12,24,36 and 48 h,respectively,from 4.71% of the control group to 29.8,34.7,46.2,and 30.0%,respectively(P<0.01).Although there was an increase in the percentage of smaller cells with weak FS signal(9.51,13.3,17.1,24.4%,P<0.001),the cell shows characteristic vacuolation under a microscope examination,and the cytoplasmic marginalization marked by FLICA inferred that this change resulted in the weakening of FS signal rather than the change in volume.Cells were further labeled with three reagents,FLICA,7-AAD,and Annexin V-APC.Flow cytometry was performed.Cells with 7-AAD negative signal were gated for further analysis.Results showed that R3(Ann +/FLICA-)had early apoptosis,and R1 had caspase-1-related death(Ann-/FLICA+),and this mechanism of death was related to apoptosis(Ann +/FLICA+).The timing study found that the Ann-/FLICA+ cells appeared first,and the Ann+/FLICA+ cells appeared later,suggesting that the caspase-1-related cell death mechanism could be converted to apoptosis.Further analysis showed that the FS signals of Ann-/FLICA+ were strong and the cell had a large volume,while the FS signals of Ann+/FLICA+ were weak,which also suggested that the increase of cell volume and activation of caspase-1 were early events in the cell death process.Caspase-1 activation is usually associated with the production of IL-1? and IL-18 in the process of cell death,so the expression levels of IL-1? and IL-18 genes in PSP-induced Molt-4 cells were analyzed by a real-time quantitative PCR method.Using a GADPH gene as an internal reference,it was found that 20,40,80 ?g/mL PSP could up-regulate the expression of IL-1? and IL-18 genes in a concentration and time-dependent manner.IL-1? was increased by up to 2.9 times(80 ?g/mL,12h),and IL-18 increased by 14.5 times(80 ?g/mL,24h).Furthermore,a DCFH-DA reagent was used to label the production of intracellular reactive oxygen species(ROS),and it was found that 20,40,80 ?g/mL PSP increased the relative fluorescence intensity of ROS positive cells by 1.2,2.7 and 3.2 times.In conclusion,PSP can increase cell volume as well as inducing apoptosis,activate caspase-1,and up-regulate the expression of IL-1? and IL-18 genes.This mechanism may be related to Pyroptosis. |
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