Inhibition Of Tetramethylpyrazine On ABC Family Members In Multidrug Resistant Human Hepatocellular Carcinoma Cells

Partâ… Establishment of ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADM1.Establishment of ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADMMethods:High-dose pulse therapy combined with continuous stepwise exposure to adriamycin(ADM) to induce resistant HCC cell line BEL-7402/ADM was conducted. The parental human hepatocellular carcinoma cells BEL-7402 was cultured,digested, and washed,and then cultured at the population of 2×10⁶ per culture bottle,ADM was added at the end concentration of 50nmol/L,the culture media was changed after 24 hours,washed by phosphate buffer solution(PBS),digested by 0.25%trypsin, subculture period was ensured according to the proliferation of cells.When the HCC had been induced continuously with a low concentration of ADM for a few months, the suspended and the dead cells and the media had been discarded before the fresh media without drug was added.At the time of that HCC cells were covered on the two-thirds area of the plate,the media containing 2000 nmol/L ADM was added for pulse therapy,followed by continuous stepwise exposure in the media containing 200 nmol/L ADM.The steps repeated by using 600,800,1000 and 2000nmol/L of ADM, until drug-resistant BEL-7402/ADM cells were well grown in the media with 2000nmol/L ADM.Results:HCC cell line BEL-7402/ADM was established.BEL-7402/ADM cells were clustered and scattered,with less multiangular,increased refractivity in cytoplasm,more shuttle,and more easily digested compared with BEL-7402.2.Determination of reversal index of human hepatocellular carcinoma cells /ADM by MTT(3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)Methods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were digested by 0.25%trypsin,suspended in RPMI 1640 medium supplemented with 8%FCS,then transferred in 96-well plate,for 200μL(2×10⁴ cells/mL) per well,and cells were divided into 6 groups,blank,sham,ADM,5-FU, VCR and CDDP.Blank group was RPMI 1640 medium supplemented with 8%FCS without any drug and cells;Sham group were BEL-7402/ADM cells without any drug. Each group of drugs contained different concentrations,and there were 4 wells for each concentration of drugs.The absorbance was measured with a microplate reader at a test wavelength of 490 nm,and a reference wavelength of 620 nm.The inhibition rate of drugs was calculated as IR=(1-A_drug/A_sham)×100%.The IC₅₀ value was calculated as lgIC₅₀=Xm-â… [P-(3-Pm-Pn)/4](Xm:lg C_max;â… :lg(C_max/C_submax);P:sum of effects;Pm:E_max;Pn:E_min.Reversal index(RI)=(IC_50Resistance)/(IC_50Parental)×100%..Results:The data of MTT showed that IC₅₀ value of ADM on BEL-7402 and BEL-7402/ADM were(1.47±0.28),(7.62±0.51)μmol/L,respectively;while data for 5 -FU were(4.56±0.72) and(21.53±2.47)μmol/L,respectively;VCR(8.98±0.30) and (80.27±11.22)μmol/L;CDDP(1.2±0.04) and(10.67±2.14)μmol/L,the reversal index of drugs were:ADM 5.18,5-FU 4.72,VCR 8.94,CDDP 8.89,respectively.3.Relationship between the concentration of ADM and the expression of P-gp in BEL-7402 by western blotMethods:Treated with 200,600,800,1000 and 2000 nmol/L of ADM,proteins in BEL-7402 and induced BEL-7402 cells were detected by western blot,andα-tublin as a reference.Blots were then exposed to a computer scanner and detected by ImageJ 1.38x(NIH.USA).Data presented are mean±SD values compared with the percentage of the level of P-gp/α-tublin in parental group(control).Results:The P-gp expression in well-cultured cells BEL-7402 in media containing 200,600,800,1000 and 2000 nmol/L ADM were(99.37±1.63),(112.36±1.59), (105.01±1.93),(102.12±1.16) and(102.84±2.18)%compared with parental groups (the ratio of P-gp/α-tublin of parental calculated as 100%),respectively,in which the expression of P-gp in 200nmol/L ADM had no statistic difference from control,but that in 600,800,1000 and 2000nmol/LADM did(P<0.05).4.Analysis of cell cycle-phaseMethods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were harvested,digested,centrifuged and washed.1×10⁶ per group was fixed by adding 70%ethanol.Samples were detected using 250μg/mL propidium iodide by flow cytocemetry.Results:Analysis of cell-cycle phase distribution was carried out on BEL-7402 and BEL-7402/ADM cells.The results showed that BEL-7402/ADM cells appeared lower value of G₀/G₁ and higher S and G₂/M(P<0.01) than that of BEL-7402.5.Intracellular accumulation of Adriamycin(ADM) in BEL-7402 and BEL-7402/ADM cells by flow cytometry(FCM)Methods:To assess the steady accumulation of ADM,BEL-7402 cells and BEL-7402/ADM cells were incubated with 8 000 nmol/L ADM for 2 h.The fluorescence intensity of intracellular ADM was recorded by FCM with an excitation wavelength(λex) of 480 nm and emission wavelength(μem) of 575 nm.Results:The mean fluorescence intensity of ADM in Resistance group was (82.08±6.89)%of Parental group(P<0.01),indicating that there was a decreased intake of ADM in BEL-7402/ADM. 6.The adherence rate and the plate cloning efficiency of BEL-7402/ADMMethods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were harvested and resuspended to be single-cell solution, respectively.The live cells were calculated on glass slide by trypan blue staining, 1×10⁶ cells per culture bottle was distributed for 6 groups,respectively.The adherent cells was calculated every 1 h as following:The adherence rate=(A_adherent/A_inoculated)×100%.Results:The data showed that the longer the time lasted,the greater the adherence rate of BEL-7402 and BEL-7402/ADM were.There was a statistic difference between the two groups at 2h,when the adherence rate of BEL-7402 was more than that of BEL-7402/ADM(P<0.05).The plate cloning efficiency of BEL-7402 cells(54.44±5.83%) was more than that of the parental cells(68.28±8.06%).Conclusion1.In this study,resistant human hepatocellular carcinoma cell line BEL-7402/ADM was established by adriamycin(ADM) using the model of high-dose pulse therapy combined with continuous stepwise exposure.The data indicated that ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADM was resistant to not only ADM,but also 5-FU,VCR and CDDP.The resistant cell line with such a phenotype would be weaker slightly after stored,but recovered and cultured for about 1 month,given induction again,the level ofphenotype would appear as before.2.The result of western blot showed that there was no effect of ADM at the concentration of 200 nmol/L on the P-gp expression in the cell line but at 600 nmol/L and more.3.As to the difference of cell cycle-phase between the parental cell line and the resistant one,the experimental results showed BEL-7402/ADM cells appeared lower value of G₀/G₁ and higher of S and G₂/M(P<0.01) than that of BEL-7402,while phase S had no change,there were differences on the adherence rate and the plate cloning efficiency between BEL-7402/ADM and BEL-7402,this result seemed to be different from other references,it maybe occur since different conditions,methods or/and deviations.4.The mechanism of the BEL-7402/ADM resistant to agents may associate with P-gp acting as a pump to the drugs according to the results of FCM and western blot.5.According to references as described previously,tumor cells are often cultured in the media containing 10%FCS,in this study,we used 8%FCS and obtained the same results,and saved a lot of agents. Partâ…¡Reversal and mechanism of tetramethylpyrazine on HCC cell line BEL-7402/ADM1.Measurement of reversal of TMP on BEL-7402/ADM by methylthiazoletetrazolium(MTT) assayMethods:To assess the reversal effect,a measurement of cells proliferation by methylthiazoletetrazolium(MTT) assay was conducted as described previously. BEL-7402/ADM cells were preconditioned with TMP(400 and 600μmol/L) or VRP (5 and 10μmol/L) in 96-well plate for 24 h,respectively,then treated with 0.3,0.6,1.2, 2.4,4.8μmol/L of ADM in 96-well plate for 72 h,respectively.Verapamil(VRP) was conducted as positive control,and then the medium was discarded and the cells were washed by PBS for 3 times.IC₅₀ value,resistance index and reversal index were calculated as described previously.The resistance index(RI) is calculated as RI= (IC₅₀ Resistance group)/(IC₅₀ Parental group)×100%.The reversal index is calculated as RI=(IC₅₀ Resistance group)/(IC₅₀ TMP/VRP group)×100%.Results:Results from the experiment showed that the reversal index were 3.79(P<0.01) by TMP at the concentration of 400μmol/L and 14.65(P<0.01) at the concentration of 600μmol/L,respectively,similar to VRP,indicating that the reversal effects of regulators took place in a dose-dependent manner.2.Intracellular accumulation of Adriamycin(ADM) by fluorescence microscopyMethods:With/without TMP(600μmol/L) or VRP(5μmol/L) preconditioning in 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2h at 37℃,washed three times with ice-cold PBS.With an excitation(^λex) wavelength of 488 nm and emission(^λem) wavelength of 575 nm,the fluorescence intensity of intracellular ADM was directly observed by fluorescence microscopy. Results:By the photos taken with fluorescence microscopy,red fluorescence was caught,increased accumulation of ADM in BEL-7402/ADM cells treated with TMP or VRP compared with that in BEL-7402/ADM cells treated without TMP or VRP, following the parental group,was observed directly.3.Intracellular accumulation of ADM in HCC cell lines by FCMMethods:With/without TMP(600μmol/L) or VRP(5μmol/L) preconditioning in 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2 h at 37℃,washed three times with ice-cold PBS.With an excitation(^λex) wavelength of 488 nm and emission(^λem) wavelength of 575 nm,the mean fluorescence intensity of intracellular ADM was recorded by FCM.Results:The mean fluorescence intensity of intracellular ADM in Parental group, TMP group and VRP group were(121.83±23.2)%(P<0.01),(163.78±39.5)%(P<0.01) and(320.1±47.18)%(P<0.01) compared to Resistance group,indicating that TMP can increase accumulation of ADM in BEL-7402/ADM.4.Intracellular accumulation of ADM by high performance liquid chromatography(HPLC)Methods:the parental and resistant cell lines were divided into Parental,Resistance, TMP(with 600μmol/L TMP) and VRP(with 5μmol/L VRP).With/without TMP or VRP preconditioning for 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2 h,washed three times with ice-cold PBS. The accumulation of ADM in cells was also quantified by the HPLC method.The linear,reproducibility,recovery,precision,accuracy,stability were observed and calculated.Results:The peak area ratio of ADM and internal standard alprazolam was obtained and calculated by HPLC.The calibration curve was obtained as the following with the content ranging from o.1 ng/mL to 2 000 ng/mL.The limit of detection(LOD) was 0.05ng/mL,and RSD was 0.407.The standard curve was C=47.73A+23.62 (correlation factor:0.99 8),where C refers to the concentration of ADM,A refers to the peak area ratio of ADM and alprazolam.The mean concentration of ADM in Parental group,Resistance group,TMP group and VRP group were (2.45±0.52),(2.06±0.40)μmol/L,(3.63±0.59) and(3.92±0.97),respectively,indicating that TMP can increase accumulation of ADM in BEL-7402/ADM as same as the result by FCM.The study also indicated that TMP increased the level of ADM without increased itself accumulation in the resistant cells,inferring TMP was not the substrate of P-gp like ADM/VRP and did not compete the ABC proteins with ADM.5.Effects of TMP on expression levels of MDR1,MRP2,MRP3 and MRP5 mRNA by real-time reverse-transcription-PCR analysisMethods:Total RNA was isolated from BEL-7402 and BEL-7402/ADM in RNA clean solution by Trizole Reagent according to the manufacturer's protocol.All primer pairs and their appropriate fluorescent hybridization probes were designed and produced by Shanghai Sangon Biological Engineering Technology and Services CO., Ltd.(China).The mRNA levels of MDR1,MRP2,MRP3 and MRP5 were measured by real-time RT-PCR and quantitated by SLAN Real-time PCR Detection System.In addition,the mRNA level of the reference geneβ-actin was measured and used to normalize the mRNA levels of the drug resistance genes.Results:The data were calculated as the expression level of mRNA of Resistance group was 100%.It was showed TMP decreased the expression of MDR1,MRP2, MRP3 and MRP5 mRNA to(0.67±0.03),(0.85±0.10),(0.90±0.04) and (0.63±0.11) compared to Resistant group(P<0.05),but this only took place on the expression of MDR1 mRNA in VRP group,as there was no statistical difference in the expression of MRP2,MRP3 and MRP5 mRNA in VRP group.6.Effects of TMP on expression levels of P-gp MRP2,MRP3 and MRP5 proteins by Western blot analysis(WB) Methods:BEL-7402 and BEL-7402/ADM cells were divided into Resistance,TMP and VRP group,and preconditioned with TMP(600μmol/L) and VRP(5μmol/L) for 3 hours,then treated with 8 000 nmol/L ADM for 2 hours.The proteins level of P-gp, MRP2,MRP3 and MRP5 were detected andβ-actin as a reference by western blot.Results:We found that compared to Resistance group,the expression of P-gp, MRP2,MRP3 and MRP5 was entirely decreased to(0.49±0.04),(0.61±0.13), (0.38±0.14) and(0.68±0.07)(P<0.01) in TMP group,however,except the expression of P-gp in VRP group,there was no statistical difference in the expression of MRP2, MRP3 and MRP5 in VRP group.Conclusion1.Tetramethylpyrazine(TMP) is a bioactive constituent isolated from the root of Ligusticum chuanxiong Hort,a Chinese herb(Chuanxiong),which can enhance the chemosensitivity effects of drug on human hepatocellular carcinoma BEL-7402 cells, acting as a MDR modulator.However,it is unknown till now if it has effect on other transporters till now.In this study,the reversal effect of TMP on MDR in multidrug resistant human hepatocellular carcinoma BEL-7402/ADM was investigated and the relationships among TMP and MDR1,MRP2,MRP3 and MRP5 were explored.It was concluded that TMP can be reverse the resistant level of BEL-7402/ADM in a dose-dependent manner.2.The Intracellular accumulation of ADM preconditioned by TMP decreased in resistant BEL-7402/ADM suggested that TMP would have effects on one and/or more ABC super family proteins.3.MDR1/P-gp,MRP2,MRP3,and MRP5 are of significance to contribute to MDR mechanism jointly in resistant HCC,and can be reversed by TMP.4.Both TMP and the first generation modulator,VRP have effects on MDR1/P-gp, TMP also has effects on MRP2,MRP3 and MRP5,but VRP has not,which deserves further study.We also found that the Intracellular accumulation of TMP in resistant cells did not alternate indicating that unlike ADM and VRP,TMP is not the substrate of P-gp/ABC family members and not resulting in competitive inhibition to ADM.