| OBJECTIVE To investigate the reversal effect of Chidamide on the resistance of human diffuse large B-cell lymphoma cell line Pfeiffer/ADM to Adriamycin,and to explore the possible mechanism.METHODS1.The Pfeiffer/ADM cell line and its parental cell line Pfeiffer were treated with different cincentrations of Chidamide or ADM alone,or in combination.The proliferation inhibition of these agents and the half maximal inhibitory concentration of ADM,Chidamide were determined by cell counting kit-8(CCK8).2.The cell apoptosis of Pfeiffer/ADM cells that treated with different concentrations of drugs was analyzed by flow cytometry.3.The mRNA expression of MDR1 of Pfeiffer/ADM cells that treated with different concentrations of drugs was analyzed by quantitative Real-time PCR.4.The histone H3,H4 acetylation levels in each group of cells treated with different doses of Chidamide were determined by Western Blot.RESULTS1.Pfeiffer and Pfeiffer/ADM cells were treated with different concentrations of adriamycin(0.01,0.02,0.04,0.08,0.16?g /mL)for 48 h,the proliferation inhibition rat of Pfeiffer cell were(10.45±0.93)%,(31.10±0.98).%,(43.00±0.64)%,(60.00±0.84)%,(81.21±21.8)%,respectively.The proliferation inhibition rats of Pfeiffer/ADM cell were(11.38±1.95)%,(14.84±2.00)%,(21.28±1.75)%,(26.71±4.31)%,(35.95±2.40)%,respectively.The proliferation inhibition rat of Pfeiffer cells by adriamycin was higher than that of Pfeiffer/ADM cells and the difference was statistically significant,P<0.05.The resistance index of Pfeiffer/ADM was 8.4.2.Pfeiffer/ADM cells were treated with adriamycin(0.16?g/mL)for 24 h,48h and 72 h,the proliferation inhibition rats of proliferation were(18.45 +1.83)%,(35.95 + 2.35)%,(52.18 + 2.56)%.The inhibition was time dependent.Pfeiffer cells were treated with adriamycin(0.16?g/mL)for 24 h,48h and 72 h,the proliferation inhibition rats of proliferation were(46.74±4.35)%,(81.21±2.20)%,(94.53±0.40)%.The proliferation inhibition rat of Pfeiffer cells by adriamycin was higher than that of Pfeiffer/ADM cells and the inhibition was time dependent.The difference was statistically significant,P<0.05.3.Pfeiffer/ADM cells were treated with adriamycin(0.16?g/mL)for 24 h,48h and 72 h,the proliferation inhibition rats of proliferation were(18.45 +1.83)%,(35.95 + 2.35)%,(52.18 + 2.56)%.The inhibition was time dependent.Pfeiffer cells were treated with adriamycin(0.16?g/L)for 24 h,48h and 72 h,the proliferation inhibition rats of proliferation were(46.74± 4.35)%,(81.21 ±2.20)%,(94.53±0.40)%.The proliferation inhibition rat of Pfeiffer cells by adriamycin was higher than that of Pfeiffer/ADM cells and the inhibition was time dependent.The difference was statistically significant,P<0.05.4.Pfeiffer cells were treated with Chidamide(8mmol / L)for 24 h,48h and72 h,the proliferation inhibition rats of proliferation were(40.57±10.00)%,(66.57±8.82)%,(91.87±4.97)%.Pfeiffer/ADM cells were treated with Chidamide(8mmol / L)for 24 h,48h and 72 h,the proliferation inhibition rats of proliferation were(20.89±2.26)%,(34.07±4.29)%,(50.50±2.21)%.The inhibition was time dependent,the difference was statistically significant,P<0.05.5.The proliferation of Pfeiffer/ADM cells which treated with different concentrations of Chidamide(0,4,8,16mmol/L)plus Adriamycin(0.08?g/ml)for 48 h were(16.91±1.78)%,(48.21±1.35)%,(53.33±2.08)%,(55.23±3.10)%,respectively.The proliferation of Pfeiffer/ADM cells which treated with different concentrations of Chidamide(0,4,8,16mmol/L)plus Adriamycin(0.16?g/ml)for 48 h were(23.17±2.58)%,(50.58±1.59)%,(56.37±1.82)%,(59.50±2.66)%,respectively.The proliferation inhibition rat of Pfeiffer/ADM cells which treated with different concentrations of Adriamycin(0.01,0.02,0.04,0.08,0.16?g/ml)for 48 h were(11.38±1.95)%?(14.84±2.00)%?(21.28±1.75)%?(26.71±4.31)%?(35.95±2.40)%,respectively.It showed that Chidamide could restore the sensitivity of Pfeiffer/ADM cells to Adriamycin and its action is concentration dependent.The difference was statistically significant,P<0.05.6.After treatment with Chidamide(0,4,8,16mmol/L)combined with Adriamycin(0.08?g/ml)for 48 h,the percentage of apoptotic cells of Pfeiffer/ADM in the control group was 1.6%,the percentage of apoptotic cells of Pfeiffer/ADM in the experimental group were(5.3±0.12)%,(6.63±0.15)%,(11.13±0.21)%,(16.77±0.15)%,The difference was statistically significant,P<0.05.7.After treatment with Chidamide(0,4,8,16mmol/L)for 48 h,the level of MDR1 mRNA of Pfeiffer/ADM cells in 4,8,16 m mol/L groups can significantly reduce,the relative expression are(9271.13±944.39),(6600.83±665.04),(158.90±16.41),respectively.It shows that Chidamid can down-regulate the expression of MDR1 mRNA,and its effect is concentration dependent.The difference was statistically significant,P<0.05.8.After treatment with Chidamide(0,4,8,16mmol/L)for 48 h,the histone H3 acetylation levels of Pfeiffer/ADM cells were(116.54±0.16),(183.23±0.60),(216.21±2.09),(243.24±0.75),the histone H4 acetylation levels of Pfeiffer/ADM cells were(86.86±0.44),(162.23±1.58),(210.50±1.34),(234.35 ± 0.29),respectively.The results showed that Chidamide could increase the acetylation levels of histone H3 and H4 in Pfeiffer/ADM cells,and its effect was concentration dependent.The difference was statistically significant,P<0.05.Conclusion1.The Pfeiffer/ADM cells used in this study were moderately resistant to adriamycin.2.Chidamide can inhibit the proliferation of Pfeiffer and Pfeiffer/ADM and induce apoptosis.Its effects were does and time dependent.3.Chidamide enhances the sensitivity of Pfeiffer/ADM cells to ADM,showing drugs resistance-reversing potential.The mechanism may be related to increase the acetylation levels of histone H3 and H4,reduce MDR1 mRNA level. |
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