Matrine-induced γ-globin Gene Expression And Erythroid Differentiation Of K562 Cells: An In Vitro Study

Background and ObjectivesBeta-thalassemia is one of the most common somatic chromosome monogenicdiseases and a chronic hemolytic anemia that imposes greatest impact on human health,its molecular pathogenesis has been found to be related to mutation or absence of betaglobin gene that leads to the imbalance between alpha globin peptide chain andnon-alpha globin peptide chain. Maturation defect and ineffective production oferythrocytes may result from excessive alpha globin peptide chain, leading toconsequent occurrence of hemolytic anemia. Due to its worldwide distribution, highincidence and severe consequences, globin has been being one of the hottest topics inthe field of biomedicine as an important paradigm for the treatment of geneticdisorders. So far, it still remains difficult in treating beta-thalassemia major and partialbeta-thalassemia intermedia, though such preventive measures as community screening,genetic counseling and prenatal diagnosis have been taken to control the birth ofperilous fetuses. Presently, therapies for beta-thalassemia include regular bloodtransfusion with iron chelating agents, patients receiving which have a mean life-spanof about 30 years; splenectomy and partial splenic embolization, which is effective for only partial beta-thalassemia intermedia; and hematopoietic stem cells (HSCs)transplantation, a radical cure for the disease, but the widespread application of whichis restricted due to the difficulty in tissue matching, complicated transplantationprocedures and high cost. At present, the most promising treatment forbeta-thalassemia is the gene therapy, including gene correction therapy and generegulation therapy in general. Gene correction therapy is to achieve functional recoveryof the cells by repairing the defective gene with DNA recombinant techniques fortreatment of genetic disorders. Theoretically, beta-thalassemia, for example, themonogenic disease, is the most ideal model for gene correction therapy, so it has beenexpected to be the first curable monogenic disease through correction of the defectivegene at the molecular level. However, it is still in its initial stage due to sometechnological puzzles. Gene regulation therapy, in a broad sense, refers to themedication therapy for genetic disorders by regulating target gene expression. It is wellknow that an increase in gamma-globin production could ameliorate the clinicalsymptoms of patients with beta-thalassemia major, which sheds lights on studies of thepast two decades to find how to prevent or postpone the expression switch fromgamma-globin gene to beta-globin gene and how to reactivate the expression ofgamma-globin gene that tends to postnatally terminate, It is the unique gene therapythat has entered into the stage of clinical research and brings about hope for patientswith beta-thalassemia.So far, the reported pharmaceutical chemicals with high therapeutic efficacy, forexample, hydraxyurea, 5-azacytidine, recombinant human EPO and butyric acid andderivatives, have such disadvantages to certain extents as marrow inhibition, potentialcarcinogenesis, high cost and inconvenient intake, while other compounds that arebeing undergone basic experimental studies have been far from clinical application and their long-term toxicological effects are still unknown. So, it is badly needed todevelop new agents that might be able to more effectively and safely stimulategamma-globin gene. Traditional Chinese herbal remedies have gained widespreadattention from both domestic and overseas scholars due to their minor side effects andabundant resources. Therefore, many scientists are attempting to screen out moreeffective, less toxic and cheaper agents as the inductor of gamma-globin gene.Many gamma-globin gene inductors were found from antitumor agents, includingchemical compounds (e.g. 5-azacytidine, arabino-furanosyl-cytosine, hydraxyurea,tallimustine, cisplatin analogs and doxorubicin derivatives), herbs (harringtonine,meisoindigo and vinblastine) for chemotherapy of leukemia. In our study, 11 effectiveparts or simple substances of Chinese medicines that are anti-leukemic confirmed by invitro and in vivo studies are used to induce hemoglobin production of human chronicmyelogenous leukemia (CML) cell line K562, by benzidine staining. Matrine wasfirstly screened out as an inductor for erythroid differentiation of K562 cells.Meanwhile, effects of matrine on K562 cells proliferative inhibition were determinedby trypan-blue dye exclusion test and the colorimetric (XTT) assay, followed bywestern blotting and real time fluorescence quantitative RT-PCR to investigate theexpression of gamma-globin gene at protein and mRNA level, respectively. We attemptto screen out a new gamma-globin inductor for the treatment of beta-thalassemia.Methods1. Methods for proliferation inhibition and erythroid differentiation of K562 cellsby matrine1.1 Methods for proliferation inhibition of K562 cells by matrine: K562 cells werecultured in RPMI-1640 containing 10% fetal bovine serum, 100U/mL penicillin,100U/mL streptomycin and 2 mmol/L glutamine in a 5% CO₂ incubator at 37℃. 1.1.1 Trypan-blue dye exclusion test: K562 cells at the exponential growth phase wereharvested to be inoculated into the culture flask of 25 cm² at the density of 5×10⁴ cells/mL, 10mL per culture flask. According to the final mass concentration of matrine,three experimental groups were established at the matrine final mass concentrations of0.05g/L, 0.10g/L and 0.20g/L. Instead of matrine, partes aequales of RPMI-1640culture fluid and 0.5mmol/L sodium butyrate were taken as the negative and thepositive controls, respectively. Trypan-blue dye exclusion test was performed atintervals of 24 h from the time of inoculation and aliquots were removed daily for up to6 days.1.1.2 Colorimetric (XTT) assay: K562 cells at exponential growth phase wereharvested and re-suspended at a density of 1×10⁴ cells/mL. Six group including threecontrols (RPMI-1640 culture medium only for a blank, a negative and a positive withaddition of 0.5mmol/L sodium butyrate) and three experimental group (with finalmatrine mass concentrations of 0.05g/L, 0.10g/L and 0.20g/L, respectively) wereinvolved in this study. At day 3, the dosage effect of matrine on K562 cellsproliferative inhibition was recorded.1.2 Methods for erythroid differentiation of K562 cells by matrine:1.2.1 Benzidine staining: K562 cells culture procedures and experimental design werethe same as those in Trypan blue exclusion assay. Trypan blue exclusion assay wasperformed at the intervals of 24 h from the time of inoculation. Benzidine staining wascarried out only in case of the cell viability greater than 95%. The percentage ofbenzidine-positive cells (visualized as cells with blue crystals) was calculated for up to6 days.1.2.2 Wright-Gimesa staining: K562 cells of negative control group and the matrinegroup at the final mass concentration of 0.10g/L at day 4 was centrifuged respectively for the preparation of cell smears and then stained with Wright-Gimesa for subsequentmorphological observations.2. Methods for matrine-induced gamma-globin gene expression in K562K562 cells culture procedures and experimental design were performed asdescribed in the section of Trypan blue exclusion assay. Western blotting techniquesand real time fluorescence quantitative RT-PCR was used to detect levels of proteinsynthesis and mRNA expression, respectively.2.1 Methods for matrine-induced fetal hemoglobin (α₂γ₂) synthesis in K562 cellsFor dose-dependent effects of matrine, K562 cells were collected fromexperimental group at the final matrine concentrations of 0.05g/L, 0.10g/L and 0.20g/L,from the positive control group at day 4 and from the negative control group,respectively. For time-dependent effects ofmatrine, K562 cells were collected from the0.10g/L matrine group at day 3, 4 and 5, from the positive and the negative controls.The total protein was extracted from the collected K562 cells and firstly was performedprotein quantitation, taking beta-actin as the house-keeping gene. These proteinblotting results of Fetal hemoglobin (α₂γ₂) and the corresponding of beta-actin fromeach group were separted by Western blotting techniques and analyzed by Gel analysissoftware package, Gel-pro analyzer 3.1, was used to determine the gray scale of theprotein bands. The ratio of each gray scale of fetal hemoglobin band to itscorresponding gray scale of beta-actin band was used for correction of protein samplequantities. The corrected expression of the negative control was taken as "1" forcalculating relative fold of each other groups, which were used for subsequentstatistical comparisons.2.2 Methods for matrine-induced ^Gγglobin gene and ^Aγglobin gene mRNA expressionin K562 cells Quantitative of mRNA expression was performed with relative quantitationanalysis. The standard curve was used for inter-sample amplifying efficiencycorrection. All these samples were performed normalization (quantitative correction ofRNA), taking beta-actin, as the house-keeping gene. When making the standard curve,cDNA sample, reverse transcription for total RNA at high concentration, was diluted10 times by specific agent, EASY Dilution. Five Ct values of amplified gradientconcentration samples were obtained through real time fluorescence quantitativeRT-PCR. Based on the 5 points, the linear relative quantitation standard curve wasfinally constructed with Ct values of each gradient samples as the y axis and with Logvalues of corresponding original template concentrations as the x axis (y=ax+b). Fordose-dependent effects, K562 cells from all groups at day 3 were collected. Fortime-dependent effects of matrine, K562 cells from the experimental group (0.10g/L ofmatrine) at day 2, 3 and 4, positive and negative controls were harvested, respectively.The extracted total RNA was firstly identified by agarose gel electrophoresis (AGE)and ultraviolet spectrophotometry, and then by real time fluorescent quantitativeRT-PCR to regulate the sample concentration for placement of Ct values on thestandard curve. The samples to be assayed and the standard sample were spontaneouslyamplified in different tubes for respective destination gene. The Ct values of eachdestination gene mRNA and house-keeping gene mRNA were then substituted into thecorresponding standard curve. The quantified house-keeping gene mRNA was taken as"1" to calculate the relative times of each destination gene mRNA as the errorcorrected total RNA. Subsequently, the corrected expression of negative control wastaken as "1" for calculating the relative expression of other group, which were used forstatistical comparisons. Results1 Effects of matrine on proliferation inhibition and erythroid differentiation inK562 cells1.1 Results of matrine on proliferation inhibition in K562 cells by trypan-blue dyeexclusion test and XTT assay: After pretreatment with matrine, decreased K562 cellscounts have statistical significance not only at different time points (P=0.000), but atdifferent matrine concentrations (P=0.000). K562 cells were inhibited after treatedwith each matrine concentration, the more proliferation inhibit strong and the moreincreasing matrine concentrateion, matrine can inhibit proliferation of K562 cells bydose-dependent way.1.2. Result of matrine on erythroid differentiation in K562 cells:After pretreatment with different concentrations of matrine, the percentage ofbenzidine-positive cells (BZ%) of K562 cells increased statistically not only atdifferent time points (P=0.000) in a time dependent way, but at different matrineconcentrations (P=0.000) in a dose dependent way. The reault indicates that matrineinduce hematoglobin production of K562 cells by dose-dependent and time-dependentway. The peak value reached 15.7% on day 4 at 0.10g/L ofmatrine.Morphological assessment of the K562 cells stained with Wright-Gimesa: theuntreated K562 cells showed features of undifferentiated progenitor in shape, whilematrine-induced K562 cells on day 4 were characterized of erythroid differentiation.2 Matrine-inducedγ-globin gene expressions in K562 cells2.1. Matrine-induced fetal hemoglobin (HbF) synthesis in K562 cells by Westernblotting: HbF production incresed were 1.08-, 1.53-, 1.36- and 1.52- fold of K562 cellsin the presence of 0.05g/L, 0.10g/L and 0.20g/L of matrine and sodium butyrate,respectively. K562 cells which induced by 0.10g/L and 0.20g/L matrine have statistical significance relative to negative control (P＜0.01). HbF production incresed were 1.38-,1.81-, 1.54- and 1.92- fold on day 3, 4 and 5 after matrine treatment and on day 3 afterincubation with sodium butyrate, respectively, as compared to negative control (P＜0.01). Matrine-induced fetal hematoglobin synthesis of K562 cells by dose-dependentand time-dependent way. The action reached its peak of K562 cells on day 4 at 0.10g/Lof matrine.2.2 Matrine-induced accumulation ofγ-globin gene mRNA in K562 cells by real timeFQ-RT-PCR.No statistical significance of ^Aγ-globin gene mRNA expression with differentconcentration matrine and on different time points among all experimental group ascompared to negative control (P＞0.05).Accumulation of ^Gγ-globin gene mRNA expression in K562 cells were 1.40-,2.72-, 2.20- and 3.39- fold on day 3 in the presence of 0.05g/L, 0.10g/L and 0.20g/L ofmatrine and sodium butyrate, respectively. ^Gγ-globin gene mRNA expression inducedby 0.10g/L and 0.20g/L of matrine have statistical significance relative to negativecontrol (P＜0.05); Accumulation of ^Gγ-globin mRNA were 1.57-, 3.08-, 2.54- and3.45-fold on day 2, 3 and 4 in the presence of matrine and on day 3 after incubationwith sodium butyrate, respectively. ^Gγ-globin gene mRNA expression induced ondifferent time have statistical significance relative to negative control (P＜0.05);Matrine-induce K562 cells accumulate ^Gγ-globin gene mRNA in a dose-dependent andtime-dependent way. The effect reached its peak on day 3 at 0.10g/L of matrine. Theseresults suggested that matrine-induced accumulativeγ-globin gene mRNA expressionwas associated with accumulation ^Gγ-globin gene mRNA expression.Conclusions:1. Matrine (at 0.05g/L, 0.10g/L and 0.20g/L) can inhibit K562 cells proliferation and induce erythroid differentiation by increasing hematoglobin synthesis in adose-dependent and time-dependent way. The effect reached its peak on day 4 at0.10g/L of matrine.2. Matrine can induce K562 cells to synthesis fetal hematoglobin in a dose-dependentand time-dependent way. The effect reached its peak on day 4 at matrine concentrationof 0.10g/L.3. Matrine-induced accumulativeγ-globin gene mRNA expression was asscciated withaccumulation ^Gγ-globin gene mRNA expression. The effect reached its peak on day 3 atmatrine concentration of 0.10g/L.4. The induction ofγ-globin genes expression by matrine is similar to that of sodiumbutyrate, which was used as positive control.5. The study paves the experimentation base for pharmacologically mediatedupregulation expression of humanγ-globin genes and production of fetal hemoglobinas a potential therapeutic strategy in theβ-thalassaemia.Main new ideas:1. 11 kinds of effective parts and simple substances of Chinese medicines that areanti-leukemic confirmed by in vitro or in vivo studies are used to induce hemoglobinproduction of K562 cells by benzidine staining, and matrine was firstly screened out asan inducer for erythroid differentiation of K562 cells.2. 0.05g/L, 0.10g/L and 0.20g/L of matrine can inhibit proliferation and erythroiddifferentiation in K562 cells and firstly verity that matrine can induce fetal hemoglobinsynthesis.3. Firstly verity that accumulation ofγ-globin gene mRNA after treatment of humanK562 cells with matrine and was associated with the increase ^Gγ, globin gene mRNAexpression. 4. Matrine is a hypo-toxious and effective inducer ofγ-globin gene and similar tosodium butyrate.