The Effect Of CPT Combined With Melphalan On The Proliferation Of Human Multiple Myeloma RPMI8226 Cells And Its Mechanism

Objective:In this study,the multiple myeloma cell line RPMI8226 was used as the study object,and the effect of CPT and Melphalan(Mel)on the proliferation of cells was observed in two aspects:First,the single drug and CPT and Mel were analyzed.The cytotoxic effect of combination therapy on RPMI8226 cells;second,detection of related genes on the apoptotic pathway after RPMI8226 cells were co-administered with two drugs.Through this study,it is expected that CPT will expand new treatment options in the clinical treatment of multiple myeloma(MM)patients to improve the prognosis of MM patients.Methods:1.Culture and passage of 1 human multiple myeloma cell line RPMI8226Human multiple myeloma cell line RPMI8226 cultured in RPMI 8226 medium containing 10%volume fraction of inactivated fetal bovine serum(FBS),100 U/ml penicillin,and 100μg/ml streptomycin sulfate at 37°C In a 5%volumetric carbon dioxide incubator,the cells were passaged every 2-3 days until the cells were in lo garithmic growth phase for subsequent experiments.2.CCK-8 assay for the effect of CPT and Mel monotherapy on the inhibition rate of RPMI8226 cellsTake logarithmic growth phase of RPMI8226 cells and adjust the cell density t o 1.5×10~4/ml,seeded in 96-well plate In addition,7 different final concentrations of CPT(500ng/ml,125ng/ml,31.25ng/ml,7.8125ng/ml,1.9531 ng/ml,0.4883 ng/ml,0.1221 ng/ml),7 different final concentrations of Mel(37.5μg/ml,28.125μg/ml,21.0938μg/ml,15.8230μg/ml,11.8652μg/ml,8.8989μg/ml,6.6742μg/ml),At differen t time periods of 24h and 48h,10μl CCK-8 reagent was added 4h before the end of culture and placed at 37°C.The 5%CO2 incubator was incubated for 4 hours.A fter the culture was completed,the OD value was measured by a microplate reader,the cell growth curve was plotted,and the cell inhibition rate was calculated.Eac h set of experiments was repeated three times and the mean and standard deviation were taken for statistical analysis.3.CCK-8 assay for the effect of CPT combined with Mel on the inhibition ra te of RPMI8226 cellsTake logarithmic growth phase of RPMI8226 cells and adjust the cell density t o 1.5×104/ml,inoculated in 96 wells.Take the inhibition rate of 1/2 IC50 and IC50 CPT final concentration respectively and combined with 7 different Mel concentra tion gradients Co-cultured for 24h and 48h,the cell inhibition rate was calculated.Take the inhibition rate of 1/2 IC50 and IC50 final concentration of Mel The cell i nhibition rate was calculated by co-cultivation with 7 concentrations of CPT for 24h and 48 h,respectively.4h before the end of culture Add 10μl of CCK-8 reagen t to a 37°C,5%volume carbon dioxide incubator for 4 h.After the end,the OD value was measured by a microplate reader,and the cell growth curve was plotted to calculate the cell survival rate.Each set of experiments was repeated three time s.Take the mean and standard deviation for statistical analysis.4.Choose the best combination of two drugsChoose the CPT concentration of 1?2 IC50 and IC50 conjunction with different concentration of Mel gradients were counted at 24h and 48h to calculate the inhibit ion rate of the cells after the combination treatment.Select inhibition rate of the 1/2 IC50 and IC50 of Mel combined with different concentration of CPT were worde d in RPMI8226 cells 24h and 48h,calculate the inhibition rate after combination th erapy.Calculation Results Using Compusyn Software to assess relationship between inhibition rate of RPMI8226 cells and combination index(CI),judgement of drug I nter-actions:CI<1,judged as 2 drugs with synergistic effect;CI=1,judged as2 drugs with superposition With;CI>1,then 2 drugs have antagonistic effect.5.Flow Cytometry assays the effect of CPT combined with Mel on the apopt osis rate of RPMI8226 CellsAccording to Compusyn software calculations,the concentration of CPT and M el with the lowest CI were used for apoptosis rate and the rate of necrosis are det ected.Take logarithmic growth phase RPMI8226 cells,adjust the cell density to 1×106/ml,inoculate at 24-well culture plate.Place control group,CPT drug group,M el drug group,CPT+Mel group,place Incubate at 37°C in a 5%CO2 incubator f or 0,2,4,and 6 hours.After the culture is completed,each group of cells is coll ected and After centrifugation in PBS,the supernatant was discarded and 10μl An nexin V-FITC conjugate and 10μl PI staining solution were added.Placed in the d ark at room temperature for 15 min,and checked by an up-flow cytometer.Each s et of experiments was repeated three times,taking the mean and Standard deviation s are statistically analyzed.6.The Realtime quantitative-PCR(RQ-PCR)method was used to examine the effects of CPT and Mel alone and in combination on the expression of apoptosis-re lated genes in RPMI8226 cells.The CPT concentration and Mel concentration at the lowest CI were used to detect relevant genes in the apoptotic pathway.Take logarithmic growth phase RPMI8226 cells,adjust the cell density to 1×106/ml,and inoculate in a 24-well culture plate.The control group,CPT single drug group,Mel single drug group,and CPT+Mel group were set and placed in a 37°C,5%CO2 incubator for 2,4,and 6 hours.After completion of culture,total RNA was extracted using TRIzol method using M-Mu Lv(TaKaRa)reverse transcriptase to obtain cDNA.Reverse transcription reaction conditions:pre-denaturation at 95°C for 2minutes,denaturation at 94°C for 30 seconds,annealing at 58°C for 30 seconds,extension at 72°C for 30 seconds,circulation for 35 times,and extension at 72°C for 10 minutes.Real-time fluorescence quantification.The PCR reaction system consisted of 2μl of cDNA(10×dilution),0.8μl of upstream primer(20μmol/L),0.8μl of downstream primer(20μmol/L),10μl of SYBR Premix Ex TaqTMII,6μl of ddH2O,and 0.4μl of ROXII.anti-Conditions:pre-denaturation at 95°C for 10 minutes,denaturation at 95°C for 15 seconds,annealing at60°C for 60 seconds,circulation for 35 times,extension at 72°C for 10 minutes.The dissolution curve was plotted and the final data was analyzed with 2-ΔΔCT.Results:1.The results of cell proliferation experiments showed that CPT inhibited the proliferation of RPMI8226 cells in a time-and dose-dependent manner.The difference in inhibitory rate among the seven concentrations of CPT was statistically significant(P<0.001),between the same oncentration for 24h and The difference in inhibition rate at 48h was statistically significant(P<0.001).The IC50 values of CPT single-drug treatment RPMI8226 cells at 24 h and 48 h were 4.839 ng/ml and 2.606 ng/ml,respectively,and the1/2 IC50 was 2.42 ng/ml at 24 h.Mel inhibited the proliferation of RPMI8226 cells in a time-and dose-dependent manner.There was a statistically significant difference in the inhibitory rate among seven concentrations of Mel(P<0.001),and there was a statistically significant difference in the inhibitory rate between 24h and 48h in the same concentration.Significance(P<0.001).The IC50 of RPMI8226 cells treated with Mel for 24 h and 48 h was 12.877μg/ml and 10.026μg/ml,respectively,and 1/2 IC50 was 6.4385μg/ml at 24 h.2.The results of cell proliferation experiments showed that when CPT was tre ated with 1/2 IC50 and IC50(2.42 ng/ml,4.839 ng/ml)and RPMI8226 cells in co mbination with seven concentrations of Mel respectively,the inhibitory rate of the c ombined drug group was lower than that of the Mel single drug group.Significantl y higher,the difference was statistically significant(P<0.001).Mel was treated wit h 1/2 IC50 and IC50(6.4385μg/ml,12.877μg/ml)and RPMI8226 cells with differ ent concentration gradients of CPT.The inhibitory rate was significantly higher in t he combination group than in the CPT group.The difference was statistically signif icant.(P<0.001)3.The combination drug index was analyzed by CompuSyn software.The resu lts showed that when the Mel of 6.4385μg/ml and 12.877μg/ml was combined wi th the concentration of CPT below 31.25 ng/ml,the CI value was less than 1 and the apoptotic rate of the combined group was signgficiant higher than the single gr oup.and the difference was statistically significant(P<0.05).When the CPT conce ntrations of 2.42ng/ml and 4.839ng/ml were combined with the concentrations of M el below 21.09μg/ml,the CI values were all less than 1,and the apoptotic rate of the combination group was higher than that of the single drug group.The differenc e was statistically significant(P<0.05).The CI value of 4.839 ng/ml CPT combine d with 6.6742μg/ml Mel was the smallest(CI=0.472),and the inhibition rate was increased from 50%and 20%to 65.95%when treated with single drug,respectivel y.In general,when the two drugs were used together at lower concentrations,the s ynergistic effect was stronger.Even when used in combination at low concentration s,the two drugs had higher inhibition rates than the single drug treatment group.4.The two lowest concentrations of CI(CPT 4.839 ng/ml and Mel 6.6742μg/ml)were selected for detection the rate of apoptosis and necrosis.The results show ed that compared with the single drug group,the positive rates of Annexin-v+/PI-a nd Annexin-v+/PI+in the combination group increased gradually with the prolongati on of drug action time,and were higher than those of the single drug treatment gr oup.The positive rate of Annexin-v-/PI+remained basically unchanged.The apopto sis rates of the control group,Mel monotherapy group,CPT monotherapy group,an d CPT combined Mel group were(2.0±1.45)%,(6.7±2.13)%,(22.2±2.04)%,(19.4±1.23),respectively.The necrosis rates were 1.8±1.69)%,(1.7±2.08)%,(2.3±1.35)%,an d(2.3±2.47)%,respectively.The apoptosis rates of control group,Mel monotherapy group,CPT monotherapy group,CPT combined Mel group for 2 hours were(4.2±2.32)%,(20.1±1.31)%,(23.4±1.68)%,(21.6±2.34),respectively.The necrosis rates wer e(4.0±1.49)%,(2.2±2.39)%,(3.4±1.93)%,and(2.3±1.93)%,respectively.The apopto sis rates of the control group,CPT monotherapy group,Mel monotherapy group,an d CPT combined Mel group at 4 hours were(6.7±1.23)%,(22.5±2.41)%,(28.2±3.31)%,(32.5±3.17),respectively.The necrosis rates were(1.7±0.97)%,(2.0±2.15)%,(2.8±1.37)%,and(4.0±1.15)%,respectively.The apoptotic rates of the control group,CPT monotherapy group,Mel monotherapy group,CPT group and Mel group were(20.1±1.72)%,(28.2±2.05)%,(37.4±1.35)%,(48.6±1.55),respectively.The necrosis ra tes were(2.2±1.97)%,(1.6±1.89)%,(5.4±1.14)%,(6.6±1.02)%,respectively.The apo ptosis rate of the combination group was significantly higher than that of the single drug group(P<0.001).The necrosis rate in the combination group was similar to that in the monotherapy group.There was no significant difference in the necrosis r ate at 6 hours(P<0.05).5.The concentration of the two drugs with the lowest CI(CPT 4.839 ng/ml and Mel6.6742μg/ml)was selected and the expression of related genes in the apoptotic pathway after single drug treatment and after the combination of the two drugs was detected by RQ-PCR.The results showed that CPT can increase the expression of DR4,DR5,and Bax genes in the cells,and at the same time down-regulate the expression of Mcl-1 genes;Mel can increase the expression of P53,DR5,and Bim genes in the cells,and simultaneously inhibit the expression of Bcl-2 and Mcl-1 genes.The relative expression of P53,DR4,DR5,Bim and Bax genes increased significantly after the combined effect of two drugs for 6h,and the relative expression of Bcl-2 and Mcl-1 genes was significantly lower than that of the single drug group.Conclusion:1.CPT combined with Mel can inhibit the proliferation of RPMI8226 cells and the two drugs had synergistic effects at low concentrations.2.The main mechanism of CPT combined with Mel inhibiting RPMI8226 cell proliferation is accelerating the apoptosis.3.CPT combined with Mel can synergistically downregulate the expression of Mcl-1and Bcl-2 genes while upregulate the expression of P53,DR4,DR5,Bim and Bax,thereby inducing apoptosis of RPMI8226 cells.