Study Of Effect In Proliferation And Apoptosis Of U266and RPMI8226Cell Lines Treated By Decitabine And Dexamethasone

Objective: Multiple myeloma (MM) is a hematopoietic malignanttumor which is derived from plasma cell, and still belongs to the incurabledisease. In the past10years, the methylation drug Decitabine (DAC) andcombined with other drugs in treatment MM being paid more and moreattention.Decitabine (DAC) is specific DNA methyl transferase inhibitor, it canreverse the methylation process of DNA and reactive silent tumor suppressorgenes. Our previous studies have confirmed that DAC could induce theapoptosis of U266and RPMI8226cell lines. Glucocorticoid has definitetherapeutic effect in the treatment of MM, it could induce the apoptosis ofMM cells. In order to further confirm the mechanism of proliferation andapoptosis induced by DAC combined with traditional drugs in MM cells, thisexperiment choose the methylation drugs DAC and dexamethasone (DEX)effect on U266and RPMI8226cell lines, to detect the expression changes ofthe DNA methyl transferases DNMT3AmRNA and tumor suppressor geneP16mRNA,Thus to provide a theoretical basis for the treatment of multiplemyeloma new.Methods:1cell line selection and culturedSelecting the IL-6dependent cell line U266and the IL-6independent cellline RPMI8226whose survival states were corresponded to the developmentalstages of tumor cell. The conventional method to cultivate U266andRPMI8226cell lines. The U266cells were half passed every72hours, and theRPMI8226cells were half passed every48-72hours, Logarithmic growth cellswere used in the experiment. Each experiment was repeated three times. 2CCK-8method to detect DAC, DEX alone and combination of the twoeffects influence the proliferation of U266and RPMI8226cell linesDAC(final concentration of0.5mmol/L), DEX (final concentration of0.0001mmol/L,0.001mmol/L,0.01mmol/L) and DAC (0.5mmol/L) combinedwith DEX (0.0001mmol/L,0.001mmol/L,0.01mmol/L) were applied to theU266and RPMI8226cells for24h,48h,72h, Observing the inhibitory rate ofcell proliferation. Computational formula: Cell proliferation inhibition rate (%)=(1-OD value of experimental group/OD value of control group)×100%. AndJin Zhengjun formula calculate combination is synergy or antagonist role.Computational formula: Q=EA+B/(EA+EB-EAEB). EAand EBis respectivelyinhibition rate of each drug, EA+Bis the share inhibition rate of the two drugs.Q quartile is more than0.85and less than1.15for adding role, Q quartile1.15for synergy, or0.85for antagonist role.3AnnexinV/PI double staining to detect apoptosisDAC (final concentration of0.5mmol/L), DEX (final concentration of0.0001mmol/L,0.001mmol/L,0.01mmol/L) and DAC (0.5mmol/L) combinedwith DEX (0.0001mmol/L,0.001mmol/L,0.01mmol/L) were applied to theU266and RPMI8226cells, Observing the apoptosis rate and the percentage ofapoptosis after was roled for24h,48h,72h.4Real-time PCR method to test the expression of P16mRNA and DNMT3-AmRNA after DAC and DEX function in U266and RPMI8226cells for48h.Extract total RNA, reverse transcript cDNA, real-time PCR method testthe expression of P16mRNA and DNMT3AmRNA. Record each target geneand reference gene CT value, calculation formula F=2-△△CT,△△CT value=(objective gene CT value of Text group-reference gene CT value of Textgroup)-(objective gene CT value of Control group-reference gene CT value ofControl group), calculate the relative expression amount. The primersequences as following: P16: upstream sequences:5’-TTCCTGGACACG-CTGGTGGTGCTGC-3’; downstream sequences:5’-CGCGGCATCTATGCG-GGCATGGTTA-3’; Fragment length:177bp. DNMT3A: upstream sequences:5’-TATTGATGAGCGCACAAGAGAGC-3’; downstream sequences:5’-GGG- TGTTCCAGGGTAACATTGAG-3’; Fragment length:138bp. β-actin:upstream sequences:5’-CCTCTGACTTCAACAGCGACAC-3’; downstreamsequences:5’-TGGTCCAGGGG TCTTACTCC-3’; Fragment length:174bp.5Statistical analysisThe software of SPSS16.0was used. Firstly make the Test of Normality.Mean±standard expresses deviation grouped data. Carry out the Homogeneityof variance test, if P>0.1, think the Variance homogeneity, then thecomparisons between two groups use t test, One-way analysis of variance wasused for comparing means in groups more than two; If P≤0.1, think thevariance heterogeneity, use the Nonparametric test. P<0.05was indicatedstatistical significance.Results:1The proliferation effect of DAC and DEX alone or combination of the twodrugs on U266and RPMI8226cells.1.1Effect of DAC alone on U266and RPMI8226cells. The inhibition rate ofU266cells after treated by DAC in24h,48h,72h increased from7.03±1.99(%)to52.12±3.36(%), the inhibition rate of RPMI8226cells increased from10.32±2.36(%) to68.75±4.85(%). Take the time as the variable of statisticalanalysis, the role of DAC on the two cell lines showed a time dependence (P＜0.05).1.2Effect of DEX alone on U266and RPMI8226cells. Differentconcentration DEX acted on U266and RPMI8226cells for24h,48h,72hrespectively. One-way analysis of variance was used to analyze data, showthat the DEX inhibited U266and RPMI8226cells proliferation, and with thetime, dose dependent, The proliferation inhibition rate increased obviously (P＜0.05).1.3Effect of DAC combined with DEX on U266and RPMI8226cells. DACcombined with DEX (0.0001mmol/L,0.001mmol/L,0.01mmol/L) acted onU266and RPMI8226cells for24h,48h,72h respectively, the proliferationinhibition rates were significantly higher than the single medicine group(P<0.05). And the proliferation inhibition rate increased with the concentration of DEX and the reaction time. The inhibition rate of U266cells increased from13.58(%)±1.87(%) to79.15±2.64(%); the inhibition rate of RPMI8226cellsincreased from18.77±2.27(%) to92.30±2.26(%); the comparison amonggroups have statistical significance (P<0.05) except between the group ofDEX (0.001mmol/L) combined with DAC and DEX (0.01mmol/L) combinedwith DAC acted on RPMI8226cells after72h have no statistical significance(P<0.05). Through the Jin Zhengjun formula calculation, the Q values werebetween0.85-1.15, illustrated the two drugs has synergy. Suggest that DACcombined with DEX could enhance U266and RPMI8226cell lines growthinhibitory effect. The combined effects of the proliferation inhibition effect ismore significant in the lower concentration of DEX.2Effect of DAC and DEX on the apoptosis of U266and RPMI8226cellsAfter AnnexinV/PI double staining, the effect of DAC alone acted onU266and RPMI8226after24h,48h,72h, compared with the control group,the apoptosis rate was significantly increased (P<0.05). The apoptosis rate ofU266cells increased from11.97±3.39(%) to35.33±5.80(%), the apoptosisrate of RPMI8226cells increased from13.36±1.25(%) to41.76±1.47(%). Italso showed that DAC mainly induced early apoptosis of both the two celllines, showed a time dependence (P<0.05). DAC combined with DEX,compared with the single drug group, the apoptosis rate was significantlyincreased (P <0.05), and the main were early apoptosis. It also showed a timedependence. The apoptosis rate of U266cells could be as high as75.20±1.70(%)，and of RPMI8226cells could be as high as86.28±2.60(%).Compared the apoptosis rate between two kinds of cells, it showed DACcombined with DEX can inhibit IL-6independent MM cell line RPMI8226rather than IL-6dependent MM cell line U266. The comparison among groupswere statistically different (P<0.05) except between the group DEX0.001mmol/L combined with DAC and the group DEX0.01mmol/L combinedwith DAC on U266and RPMI8226cells for72h (P>0.05), the results suggestthat the two drugs combination of synergistic apoptosis. Apoptosis inhibitoryeffect is more significant while DEX in lower concentration. 3Influence of DAC and DEX on the P16mRNA and DNMT3AmRNAexpressionReal time PCR results showed that DAC acted on U266and RPMI8226cells after48h, compared with the control group, the relative expression levelof P16mRNA was significantly increase, the relative expression level ofDNMT3AmRNA was significantly declined, the difference between groupswas statistically significant (P<0.05). It suggested DAC enhanced the express-ion of P16mRNA and reduce the expression of DNMT3AmRNA on U266andRPMI8226cells. Different concentrations of DEX acted alone respectively,compared with the control group, there were no significant differencesbetween the groups (P>0.05), suggested that DEX had no influence on theexpression of the P16mRNA and DNMT3AmRNA. With the combination ofDAC and DEX, compared with the group of DAC acted alone, there were nosignificant differences between the groups (P>0.05), suggested that DEXcould not enhance the effect of DAC.Conclusions:1DAC and DEX can inhibit the proliferation of U266and RPMI8226cell lines significantly, and promotes cells apoptosis, especially promotes cellsearly apoptosis, in time and dose dependent. The two drugs are synergy.2DAC combined with DEX can inhibit IL-6independent MM cell lineRPMI8226rather than IL-6dependent MM cell line U266, and Showed acombined additive effect at lower DEX concentration.3DAC can reduce the amount of DNMT3AmRNA expression, increasethe amount of P16mRNA expression, DEX has no such effect. Combinedeffect compared with DAC alone had no obvious change.4For U266and RPMI8266cells, compared with lower-dose DEX, thehight-dose DEX showed no advantage in inhibiting proliferation andpromoting apoptosis.