| Objective:To study the effects of Chidamide and Valproic acid on the proliferation and apoptosis of multiple myeloma cell lines U266 and RPMI8226.To explore the effect of Chidamide and Valproic acid on NF-?B pathway of multiple myeloma cell lines U266 and RPMI8226 in a short-term(6 h).Methods:Effects of chidamide and valproic acid on multiple myeloma cells Proliferation and apoptosis.U266 and RPMI8226 cells were treated with different concentrations of Chidamide and VPA for 6h and 48 h,CCK-8 was used to detect the proliferation,and flow cytometry was used to detect the rate of apoptosis in two cell lines.Study on the mechanism of short-term effect of NF-?B pathway on chidamide and valproic acid against myeloma cells U266 and RPMI-8226.U266 and RPMI-8226 were treated with Chidamide and VPA for different time 0h,2h,4h and 6h respectively.Western blot was used to detect the expression level of I?B,NF-?B p65 and P-IKK.Results:Part 1 CCK-8 detected cell proliferationChidamide was applied to U266 cell group at concentrations of 0 ?M,1 ?M,2 ?M,4 ?M,and 8 ?M for 6 hours,and cell proliferation inhibition rates were 0.80±0.64,-0.04±0.51,0.09±3.19,1.26±0.95,and 0.55±0.48,.There was no significant difference in the inhibition rate of cell proliferation between the experimental group and the control group(p>0.05).The inhibition rates of cell proliferation at 48 h were 5.31±3.78,23.24±5.46,29.26±3.63,65.59±1.78,and 80.08±1.05,.Statistically significant differences were observed between each experimental group and the control group(p<0.05).The same concentration gradient Chidamide was applied to RPMI8226 cells for 6h,The inhibition rates of cell proliferation were 2.56±3.05,2.4±3.21,-2.06±4.9,0.81±1.54,and-0.13±1.75,respectively.There was no significant difference between each experimental group and the control group(p>0.05).The inhibition rates of cell proliferation at 48 h were 11.25±6.76,26.63±6.00,43.84±4.33,66.37±2.17,and 83.19±1.94.Statistically significant differences were found between the experimental group and the control group(p< 0.05).Different concentrations of VPA(0.5 mM,1 mM,2 mM,4 mM)acted on U266 cells for 6 h,and cell proliferation inhibition rates were-0.56±3.12,-0.26±0.57,-0.25±1.39,and 0.39±0.98.There was no significant difference of cell proliferation inhibition rate between the experimental group and the control group(p>0.05).The inhibition rates of cell proliferation at 48 h were 7.64±5.02,35.85±3.61,80.69±1.20,and 95.87±0.53,respectively.Statistically significant were seen between experimental groups and the control group(p<0.05).Different concentrations of VPA(0.25 mM,0.5 mM,1 mM,2 mM,4 mM)acted on RPMI8226 cells for 6 h,and cell proliferation inhibition rates were 0.05±1.05,0.53±0.59,0.28±0.85,0.58±0.89,and 0.76±1.26,respectively.There was no significant difference between the experimental group and the control group(p>0.05).The inhibition rates of cell proliferation at 48 h were 28.27±3.57,46.10±2.51,67.14±3.13,83.92±0.17,and 92.18±0.66.The difference between the experimental group and the control group was statistically significant(p<0.05).Part 2 Flow cytometry detected cell apoptosisChidamide was applied to U266 cell group at concentrations of 0 ?M,1 ?M,2 ?M,4 ?M,and 8 ?M,and the apoptosis rate was 8.23±0.62,8.61±0.47,8.64±0.50,8.53±0.64,and 8.57±0.5 after 6 hours of treatment.There was no significant difference between each experimental group and the control group(p>0.05).After 48 hours,the percentage of apoptosis was 9.77±2.25,15.01±3.72,17.57±3.11,21.58±3.01,and 32.50±4.32,Chidamide(2?M,4?M,8?M)group was statistically different from the control group(p<0.05).In the RPMI8226 cell group,the concentration gradient was the same as above,and the apoptosis rate after 6 hours was 8.20±0.50,8.50±0.20,8.16±0.88,8.23±0.10,8.36±0.40.There was no statistical difference between each experimental group and the control group(p>0.05).After 48 hours,the percentage of apoptosis was 8.98±0.91,13.05±0.68,17.99±1.86,36.19±2.49,and 51.92±4.08.There was statistical difference between each experimental group and the control group(p<0.05).VPA was applied to the U266 cell group at concentrations of 0 mM,0.5 mM,1 mM,2 mM,and 4 mM,and the apoptosis rate was 7.56±0.99,7.87±0.64,8.10±0.62,8.20±0.97,8.38±0.56 respectively after 6 hours of action.There was no statistical difference between each experimental group and the control group(p>0.05).After 48 hours,the percentage of apoptotic cells was 10.81±3.4,18.93±3.25,30.04±5.85,61.32±13.81,and 63.25±12.82,respectively.The apoptosis rate of VPA(1mM,2mM,4mM)group was significantly different from the control group(p<0.05).In the RPMI8226 cell group,the concentrations were 0 mM,0.25 mM,0.5 mM,1 mM,and 2 mM,and the apoptosis rate was 8.03±0.28,8.13±0.72,7.58±0.64,8.09±0.46,8.00±0.46,after 6 hours of action.There was no statistical difference between each experimental group and the control group(p>0.05).After 48 hours,the percentage of apoptosis was 8.71±0.96,15.76±1.55,22.59±3.61,39.37±0.98,and 68.49±6.52.There was statistical difference between each experimental group and the control group(p<0.05).Part 3 Western Blot was used to detect the expression of NF-?B pathway-related proteins in the short-term after treatment with Chidamide and VPA on U266 and RPMI8226 cells.After single-agent Chidamide 2 ?M or VPA 1 mM acted on U266 cell line 0h,2h,4h and 6h respectively,the expression of I?B? increased gradually with the prolongation of drug action time,Conversely the expression levels of NF-?B P65 and P-IKK?/? gradually decrease.For RPMI 8226,after the same time as the single-agent Chidamide 2 ?M or VPA 0.5 mM,the expression of I?B?,NF-?B P65 and P-IKK?/? was consistent with the U266 expression trend.Conclusions:1.Chidamide and Valproic acid have little effect on the proliferation and apoptosis of multiple myeloma cells in a short time(6h),but can significantly inhibit the proliferation of myeloma cells and promote apoptosis at 48 h,and the effect increases with increasing drug concentration.2.Chidamide and Valproic acid can inhibit the expression of P-IKK?/? and reduce the the activation and degradation of I?B? in a short time(2-6h),thereby down-regulate the expression of NF-?B p65 and exert its effect on blocking NF-?B pathway in multiple myeloma cells. |
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