| Hepatocellular carcinoma(HCC)accounts for 80%to 90%of liver cancers and it is one of the most prevalent carcinomas throughout the world.Traditional chemotherapy is often developed chemoresistance HCC patients.Matrine is an active component of traditional Chinese medicine(TCM)and is a promising alternative HCC drug.In this study,the therapeutic effects and the underlying molecular mechanisms of matrine on the human HCC cell lineHepG2 were investigated.High dosage of matrine(1.0 mg/mL)could significantly(P<0.05)inhibit cell proliferation by 48.39±3.32%,under which cell shrinkage and disruption were observed.Flow cytometry assay showed that the proportion of G1/G0 cells significantly increased,while that of S and G2/M cells significantly decreased after treatment of matrine for 48 h.These results indicated that cell arrest by matrine appeared.Up-regulation of the hepato-specific miR-122followed by down expression of its targetcyclin G1(CGI)gene by low concentration of matrine(0.2 mg/mL)was detected using was observed using quantitative real-time PCR,immunohistochemistry(IHC)and western blot assays.In conclusion,matrineinducescell arrest and apoptosis with recovery expression of the hepato-specific miR-122 in human hepatocellular carcinoma HepG2 cell line.IntroductionLiver cancer is a major health problem,it’s the fourth most common cause of cancer death according the World Health Organization(WHO)[1].There were 22,620 new cases and 18,160 deaths related to liver cancer in 2009 in the United States.China accounts for 53%of all liver cancer death all around the world.More than 90%of the primary liver cancer in China is hepatocellular carcinoma(HCC).It is the second leading cancer killer that mainly affects middle-aged people-those in the prime of their most productive years[2].Though liver resection is an appropriate option for HCC therapy,it only applies to small percentages of the patients with early stage of HCC[3].Traditionalchemotherapy remains an important therapeutic strategy forhuman cancers.However,most chemotherapeutic drugs for HCC treatment are cytotoxic agents with a high risk of side effects,such as adriamycin(ADM),cisplatin,5-fluorouracil(5-FU)and doxorubicin[4,5].Furthermore,chemoresistance is developed in HCC patients,which presents a major obstacle to the long-term efficacy of chemotherapeutic treatments[6].Therefore,alternative treatments should be developed to improve the efficiency of HCC therapy.Traditional Chinese medicine(TCM)is appreciated for its 5000-year-old history and still holds an important position in primary health care in China.TCM could complement Western medicine by using modern techniques,thus,increasing interest in TCM is observed in the Western world.Sophoraflavescens Ait(SF)is a widely used TCM for a series of diseases including viral hepatitis,cardiac arrhythmia and skin inflammations in China[7].The active components of SF are various alkaloids,among whichmatrine has been characterized as the major bioactive component[8,9].As an alkaloid,matrine has favorable medical value.Its antiviral activity promises the use in treatment of chronic hepatitis B[10].It was reported that intramuscular injection of matrine improvedthe clinical symptoms of chronic hepatitis B patients,recovered liver functions and changed serum conversion from positive to negative HBVDNA[11].It was also shown that matrine had antifibrosis activityto inhibit the platelet-derived growth factor and transform growth factor-beta actions in hepatic stellate cells[12].Recent studies showed that matrine is effective in inhibiting cell growth and inducing differentiation in human leukemia K562 cells[9,13,14].Matrine is also a differentiation inducer in SMMC-7721 cells[15].In human multiple myeloma cells and gastric cancer MKN45 cells,matrine could induce tumor cell apoptosis by interrupting cell-cell adhesion and inhibiting cancer metastasis[16,17].Matrine could potentially prevent tumor invasion[18,19].Consequently,matrine could be a promising alternative anticancer drug for HCC treatment.At present,matrine has been used for HCC treatment in murine[7],however,the antitumor therapeutic efficacy and the underlying molecular mechanisms of matrine with respect to the physiological and pharmacological effects on human HCC have not been well characterized.MicroRNA(miRNA)are noncoding small RNA molecule consist of 18 to 23 nucleotides.which are processed from stem-loop structure of single strand RNA by Dicer enzyme and highly conserved in evolution.miRNAs could bind completely or not to recognize 3’ UTR of target mRNA molecule.miR-122a locals at 18q21.31 of human chromosome,which is one of miRNAs secific-expression in liver and high amount as much as 70%of liver-total microRNAs,and over 66,000 copies per each hepatocyte.It is involved in intracellular biology such as differentiation,proliferation,apoptosis and so on.It is reported[3]that miR-122a was down-regulated in 70%of tissue of liver cancer or cell lines from hepatocellular carcinoma,and confirmed that miR-122a is related to hepatocarcinogenesis.It is shown that expression of miR-122a in hepatocellular carcinoma Hep3B cell line was lower than that in human normal liver epithelial cell LO2 by using miRNA chip technology to detect difference of miRNAs expression.In addtion,it is shown that expression change of miR-122a was correlated to sensitivity of hepatocellular carcinoma cell line on chemotherapy drugs,while there are no studies on the relationship between expression of miR-122a and cell cycle/apoptosis of liver cancer cells.It is reported[4]that increase expression of miR-122a in hepatoma cell lines could inhibit proliferation and promote apoptosis of hepatoma cells,of which the mechanism and target is still researched.In this study,the therapeutic effects and the underlying molecular mechanisms of matrine on the human HCC cell lineHepG2 were investigated,with respect to the inhibitory effect on cell proliferation,changes of cell morphology,induction of cell apoptosis,and expression of the hepato-specific miR-122 and its target cyclin G1(CGI)gene,and apoptosis inhibitor gene Livin and Survivin.Further more,the mechanisms of Matrine induces apoptosis of hepatoma cells were investigated and experimental evidences and clinical applications of combination therapy were provided.Materials and methodsCell line and matrine treatmentThe human HCC cell lines HepG2(American Type Culture Collection(ATCC)number HB-8064)was purchased from Cancer Research Institute of China Medical University(Shenyang,China).The cells were cultured with Iscove’s modified Dulbecco’s medium(IMDM)with 10%fetal bovine serum and gentamicin.Matrine was obtained from Xian Botany Garden(Shanxi,China),and its purity was>99%as assessed by HPLC.Matrine stock solution was prepared in ddH2O at 10 mg/mL.Log-phase growing cells were seeded at 1×105 cells/mL and exposed to matrine at concentrations ranging from 0.0(negative control)to 1.0 mg/mL for 48 h.MTT assayThe effects of matrine on cell viability was assessed by MTT assay as described previously[22].Briefly,cells were plated at a density of 3000 cells per well into 96-well plates.At the end of treatment,the supernatant was removed,and 20 μL of the tetrazolium compound,MTT,and 270 mL of fresh IMDM medium were added.After incubation for 4 h at 37℃,120 μL of DMSO was placed in each well to dissolve the tetrazolium crystals.Finally,the absorbance at a wavelength of 570 nm was recorded using a multi-well plate reader(Tecan,Maennedorf,Switzerland).Each experiment was performed four times.Results are expressed as the percentage growth inhibition with respect to the untreated cells.Microscopic inspectionDigested cell culture(3×105 cells/mL)was added to a 24-well plate(0.9 mL for each well)and incubated for 12 h.Then 0.1 mLmatrine of low(0.2mg/mL)or high concentration(1.0 mg/mL)per well was added.Cells were incubated for 48 h before observation.The cells were examined using an Olympus Ⅸ70 inverted microscopy,DVC1310 digital video camera and QED Camera with Standalone 145 software.Flow cytometry(FCM)analysisHepG2 cells at log phrase were collected at a final concentration of 2 × 105 cells/ml,and were incubated in 6-well plate for 12 h(2.7 mL for each well).Then 0.3 mLmatrine of low(0.2mg/mL)or high concentration(1.0 mg/mL)per well was used to induce the cells for 48 h.Simultaneously,0.3 mL cell culture,as negative control,was cultured for 48 h,collected,washed with PBS,and fixed with 70%ethanol,in sequence.Cells were centrifuged to eliminate ethanol,washed with PBS,and stained with propidium iodide(PI)in dark for 30 min before FCM analysis.Finally,BD FACSCalibur(BD,USA)was used to detect cell cycle.Cells were sampled using sampling software CellQuest 3.0.The proportion of cells in different phrases were quantified by ModFitLT 3.0[23].Each experiment was performed four times.Measurement of apoptosis1×106 cells were treated with various concentrations of matrine for 48 h,then collected by 12,000Xg centrifugation,cells precipitation were treated through lysis buffer(10 mM Tris,pH 7.5,400 mM EDTA,and 1%Triton X-100).The supernatant after centrifugation was added into proteinase K(0.1 mg/mL)overnight at 4oC and then with RNase(0.2 mg/mL)for 2 h at 37℃.DNA was extracted by using phenolchloroform(V/V=1:1),and was separated by electrophoresis in 1.5%agarose gel,then visualized after ethidium bromide(EB)staining.Apoptotic cells was quantitated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatenick endlabeling(TUNEL)method with BD ApoAlert DNA Fragmentation Assay kit which examines DNA strand breaks during apoptosis by using.Immunohistochemical stainingImmunohistochemical staining was performed using the highly specific affinity-purified poly clonal anti-CG1 antibody.Negative control was performed using nonimmune serum instead of the primary anti body.Briefly,the sections were washed in phosphatebuffered saline followed by preincubation with 1.5%normal goat serum in phosphate buffer within a moist chamber for 4 h at room temperature.Those sections were then incubated overnight with anti-CG1 antibody at a final concentration of 2 p,g/mL.After being washed with 6 changes of phosphate-buffered saline containing 0.02%Triton X-100 over 15 min,the slides were processed for immunostaining with the avidin-biotinylated peroxidase complex method(Vector Laboratories,Burlingame,CA)according to the user manual.The tissue sections were briefly counterstained with Mayer’s hematoxylin before mounting.Cultured cells were grown on sterile coverslips in tissue culture dishes overnight,fixed with 45%acetone/10%formaldehyde in 0.1 M phosphate buffer for 5 min,and then processed for immunohistochemical assay as described above.Quantitative real-time PCR assayThe expression of mature has-miR-122(P/N:4373151)in HepG2 cells was assayed by the Taqman MicroRNA Assays(Applied Biosystems)according to Gramantieri et al[24]with some modifications.Each sample was analyzed in triplicate.Reverse transcription reaction was done starting from 10 ng of total RNA and using the looped primers.Quantitative real-time PCR was done using the standard Taqman MicroRNA Assays protocol on the 7500 Real-Time PCR System(Applied Biosystems,USA).The 20 μL PCR included 1.33 μL reverse transcription product,1×Taqman Universal PCR Master Mix,No AmpErase UNG(P/N 4324018;Applied Biosystems),0.2 μmol/L Taqman probe,1.5 μmol/L forward primer,and 0.7 μmol/L reverse primer.The reactions were in a 96-well plate at 95℃ for 10 min followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min.The method named △△Ct on relative quantitation was used to determine expression levels of miRNA.The ACt was calculated while the Ct of U6 RNA was subtracted from the Ct of the miRNA of interest.The AACt was calculated as the ACt of the reference sample(non-treated HepG2)was subtracted from the △Ct of each sample.The equation 2-△△Ct was used to generate fold change.Three repeats of reference samples were used for establishing standard curve and the△△Ct.The Taqman MicroRNA Assays for U6 RNA(RNU6B,P/N:4373381;Applied Biosystems)was used to normalize the relative abundance of miRNA.Western blot analysisCell lysates were prepared in RIPA buffer(50 mmol/L Tris-HCl buffer,pH 7.4,150 mmol/L NaCl,1%Triton X-100,1%sodium deoxycholate,and 0.1%sodium dodecyl sulfate)supplemented with 1 x Halt protease inhibitor cocktail and 1×Halt phosphatase inhibitor cocktail(Pierce,Rockford,IL).A Bio-Rad protein assay(Bio-Rad)was used to determine protein concentrations.Proteins were separated on 10-12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes(Whatman,Boston,MA).Membranes were first hybridized with specific primary antibodies and then with HRP-conjugated secondary antibodies(Cell Signaling Technology).Protein bands were visualized using a commercial Immobilon Western Chemiluminescent HRP Substrate detection reagent(Millipore,Billerica,MA).The chemiluminescence of proteins transferred to PVDF membranes was detected with ECL Plus(GE Healthcare Amersham,Piscataway,NJ).Relative protein expression values were quantitatively determined via densitometry with ImageJ software.Statistical analysisAll data were analyzed with statistical software SPSS 19.0.means±SD was shown as data.The statistical significance of the studies was determined by the parametric unpaired Student’s t test.Differences with P<0.05 are considered significant and P<0.01 are considered highly significant.ResultsGrowth inhibition of matrine on HepG2 cellsTo test the growth-modulatory effects of matrine on HepG2 cells,a wide concentration range of matrine(0.2,0.5,0.8 and 1.0 mg/mL)was used.After 48 h treatment of matrine,significant inhibition of cell growth was observed at high concentration of 0.8 and 1.0 mg/mL compared with the control groups,with an average growth inhibition rate of 44.03±4.18 and 48.39±3.32%,respectively.However,low concentration of matrine(0.2 and 0.5mg/mL)had no significant effect on cell growth in comparison with the control groups.These results indicated that the growth inhibitory effect of matrine on HepG2 cells was concentration dependent.Cellular morphological changesAfter 48 h treatment of matrine,the cell morphology was observed by FIMS.At low concentration(0.2 mg/mL)of matrine,the treated cells did not show clear difference in cellular morphology from the control groups;while clear morphological changes including cell shrinking,disruption,and destruction were observed in cells treated by high concentration of matrine(1.0 mg/mL)compared with the control group.High concentration of matrine could lead to the decrease of cell colony,which was also indicated by the degradation of cell diopter.Cell apoptosis induced by matrineTo examine the mechanism responsible for matrine-mediated cellproliferation inhibition,cell-cycle distribution was evaluated by using flow cytometry analysis.Compared with the control groups,significant(P<0.05)increase of the cell ratio of G1/G0 phrase,while significant(P<0.05)decrease of that of S and G2/M phase,was observed;most cells stayed at G1 phase after treatment of matrine at low and high concentrations(0.2 and 1.0 mg/mL).The proportion of diploid slightly increased with a decrease of aneuploid;additionally,cell debris increased largely compared with the control group.The effect of matrine on the induction of apoptosis in HepG2 cells by analyzing DNA fragmentation was also conducted.Agarose gel electrophoresis at 48 h showed that matrine treatment could result in DNA fragments in HepG2 cells.A quantitative evaluation was alsomade using TUNEL to detect broken DNA-strand.Treatment of 1.0 mg/mL matrine for 48 h induced 33.1%of apoptotic cells in HepG2 cells compared to the control cells.The results showed that a significant inhibition of cell-cycle progression in HepG2 cells which were treated with matrine,and it indicated that a clear increase of the ratio of cells in the G1 phase while compared with the control.Up-regulation of the hepato-specific miR-122 in matrine treated cellsTo study the correlation between the matrine treatment and the expression of the hepato specific miR-122,the relative expression of the miR-122 was detected among the samples.An obviously up-regulated expression of miR-122 in the matrine treated HepG2 cells was determined by quantitative real-time PCR assay.This up-regulation was further enhanced by increasing the concentration of matrine,and reached its peak,about 4.39 folds,when the matrine concentration increased to 1.0 mg/mL.Down-regulation of the miR-122 target CG1 in matrine treated cellsImmunohistochemical staining showed that almost all cell nuclei and brown cytoplasmic particles were sepia-colored after treatment of matrine at low concentration(0.2mg/mL)for 48 h.The mean OD of each group showed the expression level of the CGI gene.The mean OD of the control group was 0.5367±0.0235;that of the matrine treated group was 0.3888 ± 0.0826.The mean OD of the matrine-treated group was highly significant(P<0.01)higher than that of the control group,suggesting that low concentration of matrine was enough to inhibitCG1 gene expression.These results were also confirmed by western blot assay.The band of the control was clearly brighter(about 1.5 folds)than that of the matrine treated cells.Down-regulation of apoptosis inhibitor Livin and Survivin in matrine treated cellsqPCR and Western blot showed that both mRNA and protein expression level of Livin and Survivin downregulated when concentration of matrine upregulated while HepG2 cells were treated with matrine.In 1.5mg/mL of matrine treated group,expression of Livin and Survivin was inhibited significantly(P<0.05).DiscussionsThough traditional chemotherapy remains a main method for cancer therapy,cancer cells often develop drug resistance significantly lowering the efficiency of chemotherapeutic treatment.Furthermore,given the poor prognosis associated with some liver cancers and limited treatment options outside of surgery,patients may seek alternative treatments,including TCM products,alone or in combination with standard of care[1].Recently,the effectivity of natural products from medicinal plants used in TCM has been identified by the scientific community all over the world.More and more natural products and derivatives begin to be in the standard repertoire of cancer chemotherapy[25].Novel TCM derived anti-cancer drugs including arsenic trioxide,camptothecin,cantharidin,homoharringtonine,podophyllotoxin,vinblastine and vincristine(see reference[25]).Evidences showed that these anti-cancer TCMs function as 1)apoptosis inducer to inhibit cell growth through the genes involving in regulation of cell proliferation,angiogenesis or apoptosis[26,27];2)immune-enhancer to improve immunological function or strengthening resistance against both tumor and viruses[28].The results presented in this study suggested that matrine has an important role in suppressing tumor cell growth in the human HCC cell line HepG2.Matrine decreases the survival of HepG2 cells in a dose-dependent manner.We found that low concentration(0.2mg/mL)of matrine was sufficient to inhibit HepG2 cell growth;this cell growth arrest was concentration dependent,i.e.elevated inhibitory effect was observed when dose was increased.However,the cell morphology did not significantly change at low concentration(0.2mg/mL)until dose was increased to 1.0 mg/mL(high concentration).In accompany with cell growth arrest,HepG2 cell apoptosis was induced after matrine treatment for 48 h,as indicated by phrase changes compared with untreated control with respect to cell cycle detected by FCM assay.Significant(P<0.05)decrease of the S and G2/M phase fraction followed by increase of the cell ratio of G1/G0 phrase after treatment of matrine at low and high concentrations(0.2 and 1.0 mg/mL)was in agreement with the MTT assay.These results were.similar to the inhibitory effect of matrine on the murine H22 cell proliferation[7].Differing from the dose effect of matrine on murine H22 cells in which obvious inhibition occurred when 0.5 mg/mL matrine was used for 48 h,low concentration(0.2mg/mL)for 48 h was sufficient to induced cell apoptosis in human HepG2 cells in our study.These results indicated that low concentration of matrine inhibited HepG2 cell proliferation byretarding cell growth to prolong cell cycle.The proportion of cell apoptosis in this study was slightly low,indicating that the inhibitory effect of matrine on HepG2 cells was obtained mainly by cell cycle retardation(cell growth extension and proliferation deceleration)rather than cell apoptosis.Clear increase of G1/G0 cells and decrease of S and G2/M cells implied 268 plied that the inhibitory effect of matrine on HepG2 proliferation might be mainly attributed to cell arrest in G1 phrase.However,further studies are required to confirm the results and to investigate the underlying mechanism.The hepato-specific miR-122 is often found down-regulated in all HCC-derived cell lines[24].Using qPCR technique,the expression of the hepato-specific miR-122 was determined in the present study.Compared with the non-matrine treated HepG2 cells(control),dose-dependent up-regulation of miR-122 in all matrine-treated groups suggesting a correlation between matrine and miR-122 in the human HepG2.These clues were further enhanced by the down-regulation of CG1,a target of the hepato specific miR-122[24],by western blot assay.Low concentration of matrine(0.2 mg/mL)inhibited CG1,about 1.5 fold lower than the control,which inversely correlated with the expression of miR-122,about 1.861 folds.These results indicated that the antitumor TCM matrine could inducecell arrest and apoptosis with recovery expression of the hepato-specific miR-122 in human hepatocellular carcinoma HepG2 cell line. |
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