Berberine Inhibits Glioblastoma Through Suppressing C-Met And EGFR Cascades

Part 1:Berberine inhibits glioblastoma through suppressing c-Met and EGFR cascadesBackgroundAround the world,plants with pharmacological properties are the main medical resource in developing countries[2,3].Berberine,the major component of Coptidis Rhizoma(Huanglian),has been used for the prevention and treatment of many diseases with low toxicity in China for hundreds of years,including gastrointestinal infections,abdominal pain,diarrhea,hyperglycemia,and hyperlipidemia[4-7].Recent studies have drawn extensive attention toward its antineoplastic effect in several types of human cancer cells,such as glioblastomaand lung cancer[8,9].Berberine is able to exert in vitro anti-proliferative effect by inhibiting DNA topoisomerase I and inducing cell-cycle arrest[10].The pro-apoptotic effects of berberine could be mediated through modulation of the HER2/PI3K/AKT signaling pathway,activation of caspases,and induction of poly(ADP-ribose)polymerase-1(PARP-1)cleavage[11].In addition,some studies showed thatberberine targeted side-population(SP)cells and suppressed stem cell-associated genes suchas SOX2 and NANOG.These findings demonstrated that berberine exerts its anti-tumor effects by targeting multiple molecules in different cancer cells[12].Malignant gliomas are the most common primary tumors of thebrain and among the most aggressive human tumors[13].Currently available treatment options,such as operation,radiotherapy,and chemotherapy,can only minimallyprolong life expectancy,with most patients diagnosed with aglioblastoma usually dying within 1 year[14].Recently,some studies showed that glioblastoma multiforme(GBM),a highly malignant brain tumor of astrocytic origin,concomitantly expressed HGF/SF and c-Met.This finding suggests that c-Metmay serve as a promising target of therapeutic intervention of brain cancer[15].c-Met,encodes the high-affinity receptor for hepatocyte growth factor(HGF)or scatter factor(SF),was discovered as an oncogene in tumorigenicityassay many years ago[16].Aberrant c-Met signaling hasbeen documented in most solid tumors and hematologicalmalignancies,such as human glioma,lung cancer and mesothelioma[17].An activated c-Met promotes tumor cell growth,survival,migration and invasion,c-Met also supports the multipotency in glioblastoma stem cells[18-20].c-Met kinase activates anumber of signaling pathways,such as mitogen-activated protein kinase(MAPK),phosphoinositide-3 kinase(PI3K)and STAT3 that are well established signaling cascades responsible for the diverse process,including proliferation and survival,and metastasis[21,22].Furthermore,c-Met interacts with other cell surfacereceptors,in particular EGFR,and signals to the same downstreampathways[23].In GBM,over-expression of EGFR is common and has been the major target for GBM therapy.However,the resistance of GBM to EGFR-targeted therapy developed gradually and has yielded unsatisfactory results.A part of the reasons that contributed to the resistance were related to EGFR’s cross talk with c-Met.Therefore,it is important to target both EGFR and c-Metsimultaneously for the therapy of GBM[24].In this study,we examined the effect of berberine on glioblastoma cells.We found that berberine inhibited proliferation and induced apoptosis through downregulating expression of c-Met and EGFR signals,thereby suppressing activity of their downstream targets AKT,ERK and STAT3.Importantly,berberine inhibited tumorigenesis of glioblastoma in vivo.Taken together,our results demonstrated that berberine acts as a c-Met and EGFR dual inhibitor in suppressing glioblastoma growth and might represent an effective therapeutic agent for patients with GBM.Objectives1.Evaluate the effect of berberine on GBM.2.Explore the molecular mechanisms by which berberine inhibits GBM.3.Verify the mechanisms of berberine in others cell lines.Materials and MethodsChemicals and antibodiesBerberine hydrochloride(C2OH18CINO4)was purchased from Nanjing TCM Institute Of Chinese Materia Medica(Nanjing,China).It was dissolved in diMethyl sulfoxide(DMSO)(Sigma-Aldrich,MO,USA)to a stock concentration of 30 mg/mL and stored at 4℃ for no more than two months.Antibodies used in Western blot analyses include EGFR,p-Met(Tyr1234/1235),AKT1/2/3,p-ERK(Thr202/Tyr204)and ERK 1/2(Cell Signaling,USA),c-Myc and Cyclin D1(Abcam,USA),p-Akt(Ser473),c-Met,p-STAT3(Tyr705),p-STAT3(Ser727),STAT3 andβ-catenin(Santa Cruz Biotechnology,USA)Cell cultureAll cell lines were preserved in our laboratory.The glioma cell lines U87 and U251 were maintained in DMEM medium supplemented with 10%FBS.The lung cancer cell line NCI-H1975 was maintained in PRMI-1640 medium supplemented with 10%FBS at 37℃in a 5%CO2 atmosphere.MTT assayCells were digested and seeded in 96-well microplates at a density of 3,000 to 5,000 cells per well for 24hr.After treatment with different concentrations of berberine as indicated for 24,48 and 72hrat 37 ℃,20 μl of 5 mg/ml MTT[3-(4,5-diMethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide](Beyotime,China)was added to each well.Four hours later,100 μl of DMSO was added to dissolve the resulting formazan crystals.The absorbance density was read at the wavelength of 490 nm.EdU assayU87 and U251 were seeded in 96-well microplates at a density of 8000 cells per well for 24hr.The cells were then treated with berberine at various concentrations.After 24 hour of incubation,cells in S phase were detected by EdU(5-Ethynyl-2’-deoxyuridine)assay(RiboBio,China)according to the manufacturer’s instruction.Detection of apoptosisAll cells were initially plated at a density of 1 × 105 in 60-mm dishes.At 70-80%confluency,cells were incubated under various concentrations ofberberine for 48h.Apoptosis assay was performed by Annexin V-FITC and Propidium Iodide(PI)double staining with Apoptosis Detection Kit(BestBiot,China),,following the manufacturer’s instruction.Briefly,cells were digested,centrifuged and washed withPBS twice,resuspended with 500μl Annexin binding buffer.Then 5μl Annexin V-FITC solution was added for 5 min at room temperature.PI(5μg/ml,5μl)was added in and incubated for another 5 min.Cell suspension were analyzedwithin 1 hr by using a FACSAria flow cytometer(Beckton Dickson,FranklinLakes,NJ,USA).Neurosphere formation assay.104 tumor cells were seeded in 6-cm culture dishes,washed with PBS and plated in serum-free Dulbecco’s modified Eagle’s medium(DMEM)/F 12 supplemented with B27(Gibco,USA),epidermal growth factor(EGF)(20 ng/ml,Invirogen,USA),and fibroblast growth factor(FGF)-2(20ng/ml,Invirogen,USA).Different concentrations of berberine were added to the medium.After 7 days of culture,images of neurospheres were visualized under an inverted microscope(Nikon,Japan).Western blot analysisCells were treated with berberine at various concentrations for indicated time periods.Cell lysates were harvested by scraping the cells with RIPA Buffer(50 mM Tris-HCl pH 8.0,1 mM EDTA pH 8.0,5 mM DTT,2%SDS),supplemented with a protease inhibitor cocktail(Beyotime Inc,China)and a phosphatase inhibitor(CWbio,China),and incubated on ice for 30 minutes.Cell lysate was centrifuged at 4℃ and 12,000 rpm for 15 minutesand the supernatant liquid was further recovered.The protein concentration was determined by applying the BCA assay(Beyotime Inc,China),according to the manufacturer’s protocol.Equal amount of protein lysate(30 μg)was resolvedusing a 10%SDS-PAGE gel and subsequently electro-transferred to polyvinylidene fluoridemembranes(Bio-Rad,USA).The membrane was then probed with desired primary antibodies overnight at 4℃.Horseradish peroxidase-conjugated secondary antibodies were incubated for1 h at room temperature.Positive immunoreactions were detected by using ECL Plus Western blotting detection system(Bio-Rad,USA).Nude mouse xenograft modelNude Balb/c mice were purchased from Animal centre of Guangdong Province.A total of 1.5×106 U87 cells were inoculated subcutaneously in the flank of 4-5 week-old male nude mice.When mean tumor size reached a predetermined range,animals received either vehicle as a control or berberine treatment at 15 mg/kg body weight by i.p.injection every other day for 15 days.Tumor sizes were measured with a vernier caliper every other day andtumor volumes were further calculated as length × width2/2.The animals were sacrificed when tumor grow to the certain size.ImmunohistochemistrySubcutaneous tumors developed in nude mice were fixed in 4%paraformaldehyde,paraffin-embedded,sectioned,and mounted on slides.The sections were subjected for hematoxylin and eosin staining or for immunohistochemistry analyses,which followed standard procedures.Briefly,the sections were incubated in 10 mmol/L citrate buffer(pH 6.0)at 120℃ for 2 min,and then slowly cooled down to the room temperaturein the potfor antigen retrieval.After rinsing with PBS,the sections were treated with normal blocking serum for 30 min.Primary antibodies diluted in PBS were applied to the sections and incubated at 4℃ overnight in a humidified chamber.All antibodies were diluted according to manufacturer’s instructions.After washing with PBS,all sections were incubated sequentially with a biotin-conjugated secondary antibody for 1 hour at room temperature.Immunoreactivity to specific protein was visualized using DAB(3,39 Diaminobenzidine,Burlingame,USA)as a chromogen.Counterstaining was performed by hematoxylin before dehydration and mounting.RESULTS1.Berberine inhibited proliferation and inducedapoptosis in GBM cells.1).By performing MTT assay,we found that berberine significantlyinhibited the proliferation of two cell linesstudied in a dose and time dependent manner,with a mean IC 50 value of～32μg/mL for U87 and～20μg/mL for U251(72h).2).We further measured effect of BBR on DNA synthesis with EdU(5-ethynyl-2 ’-deoxy uridine)incorporation assay.The results showed that EdU-positive cells,that represent cells in S phase,were significantly decreased with increasing concentrations of BBR in both U87 and U251 cells.3).We next examined effect of BBR on apoptosis in GBM cells.U87 and U251 cells were treated with different concentrations of BBR for 48h.The frequency of cell death was detected using flow cytometric analysis with annexin V and PI staining.At lower dosages,BBR is only margionally effective in induction of cell death.However,both early apoptotic(annexin V+/PI-)and late apoptotic(annexin V+/PI+)cells were significantly induced in the presence of 60 μg/ml of BBR.2.Berberine inhibitedc-Met and EGFR signaling cascades in GBM cells.1).Berberine inhibitedexpression levels of c-Met and EGFR in a dose-and time-dependent manner.2).Total and phosphor-STAT3,phospho-ERK and phospho-AKT,critical downstream molecules of c-Met and EGFR,were signifcantly downregulated by berberine in a dose-and time-dependent manner in two GBM cells.3.Berberine also downregulated c-Met and EGFR signaling in lung cancer.4.Berberine exerted antitumor activity in vivo1).Berberine treatment significantly inhibited the growth of GBM xenografts in nude mice,as evidenced by the changes in tumor weights and sizes that were significantly reduced by BBR treatment.While the toxicity that berberine brought was very mild when compared with control group.2).Tumor tissue from mice treated with berberine expressed low levels of EGFR and c-Met compared with control.Conclusions1.Berberine suppressed the growth of GBM cells through inhibiting proliferation and inducing apoptosis.2.Berberine inhibits the growth of GBM by downregulating EGFR and c-Met signaling cascades in vitro and in vivo.3.Inhibitory effects of berberine on EGFR and c-Met signaling cascades were also observed in lung cancercells.Part 2:Berberine increases doxorubicin sensitivity through suppressing STAT3 in lung cancerBerberine(BBR),an alkaloid component isolated from Chinese medicinal herb Huanglian,has aroused broad interests for its anti-tumor effect in recent years.The signal transducerand activator of transcription 3(STAT3),plays critical roles in malignant transformation and progression and was found to be constitutively activated in a variety of human cancers.In this study,we show that BBR inhibited cell proliferation,induced apoptosis,and suppressed tumor spheroid formation of lung cancer cell lines.These effects were correlated with BBR-mediated suppression of both phosphorylated and total levels of STAT3 protein.Furthermore,BBR promoted STAT3 degradation through enhancing ubiquitination.Importantly,we demonstrated that BBR was able to inhibit doxorubicin(DOX)-mediated STAT3 activation and sensitize lung cancer cells to the cytotoxic effect of DOX treatment.Given that BBR is widely used in clinic with low toxicity,our results are potentially important for development of a novel combinatorial therapy with BBR and DOX in the treatment of lung cancer.