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Research article

Berberine enhances inhibition of glioma tumor cell migration = and=20 invasiveness mediated by arsenic trioxide

Tseng-Hsi Lin^*=20¹^,3 ,=20 Hsing-Chun Kuo^{* =}¹^,4 ,=20 Fen-Pi Chou¹ and = Fung-Jou Lu²

¹Institute of Biochemistry and Biotechnology, Chung = Shan=20 Medical University, Taichung 402, Taiwan

²School of Applied Chemistry, Chung Shan Medical = University,=20 Taichung, Taiwan

³Division of Hematology, Department of Internal = Medicine,=20 Taichung Veterans General Hospital, Taichung, Taiwan

⁴Department of Medical Research, Chang Gung Memorial = Hospital, Kaohsiung Medical Center, Kaohsiung 833, = Taiwan

=20 author email=20 corresponding author email* = Contributed=20 equally

BMC Cancer 2008, = 8:58doi:10.1186/1471-2407-8-58

The electronic version of this article is the complete one and = can be=20 found online at: http://www.biomedcen= tral.com/1471-2407/8/58

Received:	3 March 2007
Accepted:	25 February 2008
Published:	25 February 2008

=C2=A9 2008 Lin et al; licensee BioMed Central Ltd.
This is = an Open=20 Access article distributed under the terms of the Creative Commons = Attribution License (http://creativecommon= s.org/licenses/by/2.0),=20 which permits unrestricted use, distribution, and reproduction in = any=20 medium, provided the original work is properly cited.

Abstract

Background

Arsenic trioxide (As₂O₃) exhibits = promising=20 anticarcinogenic activity in acute promyelocytic leukemic patients = and=20 induces apoptosis in various tumor cells in vitro. Here, = we=20 investigated the effect of the natural alkaloid berberine on=20 As₂O₃-mediated inhibition of cancer cell = migration=20 using rat and human glioma cell lines.

Methods

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium = bromide (MTT)=20 assay was used to determine the viability of rat C6 and human U-87 = glioma=20 cells after treatment with As₂O₃or = berberine, and=20 after co-treatment with As₂O₃and berberine. = The=20 wound scratch and Boyden chamber assays were applied to determine = the=20 effect of As₂O₃and berberine on the = migration=20 capacity and invasiveness of glioma cancer cells. Zymography and = Western=20 blot analyses provided information on the effect of = As₂O₃₌₂₀and berberine on the intracellular translocation and = activation of=20 protein kinase C (PKC), and some PKC-related downstream factors. = Most=20 assays were performed three times, independently, and data were = analyzed=20 using ANOVA.

Results

The cell viability studies demonstrated that berberine enhances = As₂O₃-mediated inhibition of glioma cell = growth=20 after 24 h incubation. Untreated control cells formed a confluent = layer,=20 the formation of which was inhibited upon incubation with 5 =CE=BCM As₂O₃. The = latter effect was=20 even more pronounced in the presence of 10 =CE=BCM=20 berberine. The As₂O₃-mediated reduction in = motility=20 and invasion of glioma cells was enhanced upon co-treatment with=20 berberine. Furthermore, it has been reported that PKC isoforms = influence=20 the morphology of the actin cytoskeleton, as well as the = activation of=20 metalloproteases MT1-MMP and MMP-2, reported to be involved in = cancer cell=20 migration. Treatment of glioma cells with As₂O_{3 =}and=20 berberine significantly decreased the activation of PKC =CE=B1 and =CE=B5 and led to actin = cytoskeleton rearrangements.=20 The levels of two downstream transcription factors, myc and jun, = and=20 MT1-MMP and MMP-2 were also significantly reduced.

Conclusion

Upon co-treatment of glioma cells with As₂O_{3 =}and=20 berberine, cancer cell metastasis can be significantly inhibited, = most=20 likely by blocking the PKC-mediated signaling pathway involved in = cancer=20 cell migration. This study is potentially interesting for the = development=20 of novel chemotherapeutic approaches in the treatment of malignant = gliomas=20 and cancer development in general.

Background

Arsenic trioxide (As₂O₃) can effectively = induce=20 apoptosis in acute promyelocytic leukemia (APL) cells in vitro = and in vivo [1-4] = and was=20 approved by the United States Food and Drug Administration in 2000 = for the=20 treatment of patients with relapsed/refractory APL. Although=20 As₂O₃has been evaluated in clinical studies = for the=20 treatment of acute myelogenous leukemia, myelodysplastic syndrome, = and=20 multiple myeloma [5], = the diverse=20 sensitivities of different types of tumor cells to this drug = limits its=20 clinical application in a wider spectrum of hematological and, = especially,=20 solid malignancies [6-12]. = The=20 antiproliferation mechanisms of As₂O₃in = solid=20 tumors are not well known, and studies on the anti-invasive = effects are=20 rare [13]. = Most=20 recently, As₂O₃was reported to induce = apoptosis in=20 a human neuroblastoma cell line via the upregulation of caspase 3 = [14].=20 Paradoxically, arsenic compounds are well-known human carcinogens = that may=20 cause, at relatively high concentrations and/or exposures times [15], = tumors in=20 a variety of human tissues including skin, liver, and kidneys [10].

The poor prognosis of human malignant gliomas is due to their = invasion=20 and recurrence. The invasion of glioma into normal brain tissue is = a major=20 challenge to clinical intervention because these tumors often = highly=20 infiltrate the surrounding brain tissues. An important = characteristic of=20 high-grade central nervous system tumors is the presence of = massively=20 upregulated protein kinase C (PKC) when compared to normal glia [16,17]. = PKC=20 represents a family of lipid-dependent serine/threonine kinases = that=20 consist of at least 12 mammalian isoforms divided into three = subfamilies=20 including conventional or classic PKCs (cPKC), non-classic or = novel PKCs=20 (nPKC), and atypical PKCs [18]. = The=20 activation of most PKC isoforms depends on the translocation from = the=20 cytosol to subcellular compartments such as the cell membrane [19]. = The=20 enhanced PKC levels in glioma cells have been suggested to be = critical to=20 the hyper-proliferative state and the resistance to apoptosis as = well as=20 glioma invasion [20,21]. = Indeed,=20 treatment of the human glioblastoma cell line T98G with hypericin = results=20 in a significant inhibition of the cell invasion, an effect that = is also=20 obtained using specific PKC inhibitors [22], = and a high=20 level of PKC=CE=B1 expression in a = human=20 colon-adenocarcinoma cell line has been correlated with high = migratory=20 activity of colon carcinoma cells [23].=20 Consequently, specific PKC inhibition is thought to control tumor = growth=20 and development [18,24]. = PKC=CE=B1/=CE=B2 = inhibitor Go6976 blocks=20 the invasion of urinary bladder carcinoma cells [25] = and PKC=20 antisense oligonucleotide LY900003 is in clinical development as a = drug=20 against breast cancer to be used in concert with, for instance,=20 chemotherapy [26].

Tumor invasion including that of high-grade malignant gliomas = is, for a=20 significant part, mediated by the overproduction of a number of=20 tissue-digesting matrix metalloproteinases (MMPs) such as MMP-2 = (type IV=20 collagenase or gelatinase A) and their activators such as = membrane-type 1=20 metalloproteinase (MT1-MMP) [27,28]. = Inhibition=20 of MMP-2 in human glioma cell line U-87 results in a dramatic = reduction in=20 cell invasion [29], = and=20 induction of PKC activation in a D54 human glioblastoma cell line = results=20 in enhanced invasion through the activation of several = metalloproteases=20 including MMP-2 [30].

Berberine, a natural alkaloid, has been extensively studied and = shown=20 to exhibit multiple pharmacological activities such as = anti-bacterial [31],=20 anti-oxidative [32], = and=20 anti-cancer and anti-inflammation capabilities [32-34].=20 Berberine also sensitizes human glioma cells to ionizing radiation = in=20 vitro [35]. = Recently,=20 berberine has been shown to exert anti-metastatic properties in = non-small=20 lung cancer cells [36], = to be=20 cytotoxic to human tumor U937 and murine melanoma B16 cells [37], = and to=20 inhibit growth and induce G1 cell cycle arrest followed by = apoptosis in=20 human epidermoid carcinoma A431 cells [38].

Although treatment with As₂O₃induces = clinical=20 remission in patients with APL without severe toxicity, relapse = with=20 As₂O₃-resistant cells still occurs [39] = and, in=20 addition, As₂O₃remains a toxic compound at=20 relatively high concentrations or exposures [15]. = Thus,=20 alternative strategies that would enhance cellular sensitivity to=20 As₂O₃, thereby lowering its concentration of = action,=20 would be helpful. In the present study, we report for the first = time that=20 berberine enhances the antiproliferation activity of = As₂O₃₌₂₀in rat glioma and human malignant glioma cell lines. In = addition, we=20 investigated As₂O₃-mediated suppression of = glioma=20 cell invasion which likely involves PKC signaling, ERK = phosphorylation,=20 and MMP-2 activation. Thus, this study may have important clinical = applications in the design of strategies to treat human = glioma.

Methods

Cell culture

The rat C6 glioma cell line was originally derived from a=20 N-nitrosomethylurea-induced rat brain tumor [40]. = C6 rat=20 cancer cells and U-87 human malignant glioma cells were cultured = in=20 minimal essential medium and RPMI (Gibco, Carlsbad, California)=20 supplemented with 10% fetal calf serum (Gibco) and antibiotics = (100=20 units/ml penicillin and 100 =CE=BCg/ml = streptomycin)=20 at 37=C2=B0C in a humidified atmosphere composed of 5% CO_{2 =}and 95%=20 air. All experiments were performed in plastic tissue culture = flasks,=20 dishes, or in microplates (Nunc, Naperville, Denmark).

Chemical reagents and antibodies

As₂O₃was obtained from TTY Biopharm = (Taipei,=20 Taiwan). Berberine, gelatin, and = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl=20 tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. = Louis,=20 Missouri). Anti-phospho-p44/42 MAPK and horseradish = peroxidase-linked=20 anti-rabbit or mouse IgG were from Cell Signaling Technology = (Beverly,=20 Massachusetts). Anti-MT1-MMP1, MMP-2, and PKCs =CE=B1=20 and =CE=B5 were purchased from Santa Cruz Biotechnology (Santa = Cruz,=20 California). Monoclonal anti-ornithine decarboxylase (ODC) = antibody was=20 purchased from Sigma-Aldrich.

Cell growth and proliferation assay

Cell viability was determined using the MTT quantitative = colorimetric=20 assay [41]. = The cells=20 were seeded at 5 =C3=97 10⁴cells/ml density and = incubated with=20 berberine or As₂O₃at various concentrations = (0, 1,=20 2, 5, 10, and 20 =CE=BCM) for 24 h. = Thereafter, the=20 medium was changed and cells were incubated with MTT (0.5 mg/ml) = for 4 h.=20 The viable cell number is directly proportional to the production = of=20 formazan, which can be measured spectrophotometrically (=CE=BB =3D 563 nm) upon solubilization with = isopropanol. Cell=20 growth was determined by counting the number of cells at indicated = periods=20 of time using a Coulter counter and measured using the trypan blue = (0.2%)=20 exclusion assay [42].

Boyden chamber assay

The Boyden chamber assay used for the analysis of tumor cell = migration=20 is based on a chamber with two medium-filled compartments. C6 and = U-87=20 glioma cells were allowed to grow as discrete colonies and treated = with=20 As₂O₃or berberine as described above. Cells = were=20 collected by trypsinization and suspended in serum-free medium at = 1 =C3=97=20 10⁵/ml. Migration assays were carried out in a 48-well=20 chemotaxis chamber (Neuro-Probe, Gaithersburg, Maryland). The = medium=20 containing 10% fetal calf serum was added to the lower chamber. = The lower=20 and upper chambers were separated by an 8 =CE=BCm=20 pore size polycarbonate membrane (Poretics, Livermore, = California). Cells=20 were allowed to migrate for 24 h at 37=C2=B0C in a humidified = atmosphere=20 containing 5% CO₂. The membrane was fixed in methanol = for 10=20 min and stained with modified Giemsa stain (Sigma-Aldrich) for 1 = h. Cells=20 on the upper side of the membrane were removed by cotton swabs. = Cells on=20 the lower side of the membrane were counted using a light = microscope at=20 200=C3=97 magnification. The number of cells that migrated to the = lower side of=20 the membrane was determined [36].

Matrigel invasion assay

Rat C6 and human U87 glioma cells were incubated with = Dulbecco's=20 modified Eagle's medium (DMEM) in 10% fetal calf serum and then = collected=20 by trypsinization. Cells (1 =C3=97 10⁵/ml) in = serum-free medium were=20 added to an inner cup of the 48-well Transwell chamber (Corning = Life=20 Sciences, Corning, New York) that had been coated with 50 =CE=BCl of Matrigel (BD Biosciences, = Franklin Lakes, New=20 Jersey; 1:10 dilution in serum-free medium). Medium supplemented = with 10%=20 serum or indicated agent was added to the outer cup. After 24 h, = cells=20 that had migrated through the Matrigel and the 8 =CE=BCm pore size membrane were fixed, = stained, and counted=20 under a light microscope. Each experiment was performed in = triplicate.

Scratch assays

Scratch assays were performed by plating cells in 6-well = culture dish.=20 After C6 glioma cells were allowed to attach and reach confluence, = a=20 scratch (4 mm) was made through the culture dish. The cells were = washed=20 twice with phosphate-buffered saline (PBS, pH =3D 7) before their = subsequent=20 incubation with culture medium in the absence (control) or = presence of=20 As₂O₃or combinations at appropriate = concentrations.=20 Photographs of treated cells moving within the scratch were taken = at the=20 indicated time points. Openlab v3.0.2 image analysis software=20 (Improvision, Coventry, United Kingdom) was used to quantify the = area=20 progressively filled with cells over the period of the = experiment.

Preparation of total cell extracts and immunoblot = analysis

Cellular lysates were prepared by suspending 1 =C3=97 10^{6 =}cells=20 in 200 =CE=BCl of lysis buffer (137 mM = NaCl, 15 mM=20 EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl₂, 0.1% = Triton=20 X-100, 25 mM MOPS, 100 =CE=BCM = phenylmethylsulfonyl=20 fluoride and 20 =CE=BCM leupeptin, = adjusted to pH=20 7.2). The cells were disrupted by sonication and extracted at = 4=C2=B0C for 30=20 min. The supernatant was quantitated using the Pierce BCA protein=20 quantitation assay (Pierce, Rockford, Illinois) and were=20 electrotransferred to Immobilon-P membranes (Millipore, Bedford,=20 Massachusetts). Detection of specific proteins was carried out = with an=20 enhanced chemoluminescence Western blot kit [43].

Immunofluorescent staining assay

One day after plating on coverslips, the cells were treated = with empty=20 vehicle or indicated agent for appropriate times. Following the = treatment=20 period, the cells were fixed in 2% formaldehyde in PBS for 10 min. = The=20 cells were then permeabelized in PBS containing 1% bovine serum = albumin=20 and 0.1% Triton X-100 for 10 min. The cells were then incubated = for 1 h=20 with rabbit anti-F-actin (Abcam, Cambridge, Massachusetts), washed = in PBS,=20 and incubated for 1 h with anti-rabbit tetramethylrhodamine = isothiocyanate=20 (TRITC) labeled secondary antibodies (Molecular Probes, Carlsbad,=20 California). Following three additional washes, the coverslips = were=20 mounted on glass slides in anti-fade medium. The images were then=20 collected using fluorescence microscopy (Carl Zeiss, Oberkochen,=20 Germany).

PKC translocation assay

Cells were washed twice with PBS and lysed by suspension in 0.5 = ml (2 =C3=97=20 10⁶cells) of ice-cold homogenization buffer (20 mM = Tris=C2=B7 HCl=20 [pH 7.5], 2 mM EDTA, 2 mM EGTA, 6 mM DTT, 50 =CE=BCg/ml aprotinin, 48 =CE=BCg/ml=20 leupeptin, 5 =CE=BCmol/l pepstatin A, = 1 mM=20 phenylmethylsulfonyl fluoride). The homogenate was sonicated and = then=20 centrifuged for 10 min at 500 =C3=97 g. The supernatant = was=20 centrifuged at 100,000 =C3=97 g for 1 h at 4=C2=B0C. The = resulting=20 supernatant was designated as the soluble (cytosol) fraction. The = pellet=20 was solubilized in 200 =CE=BCl of = homogenization=20 buffer containing 1% Triton X-100 and centrifuged at 15,000 =C3=97 = g=20 for 15 min at 4=C2=B0C. The supernatant was designated as the = particulate=20 (membrane) fraction. All extraction procedures were performed at = 4=C2=B0C. The=20 protein concentration in cell fractions was determined using a = commercial=20 protein assay (Bio-Rad, Hercules, California) with bovine serum = albumin=20 (BSA) as a standard. Extractions for immunoblotting were based on=20 previously described methods.

Zymography

MMP-2 and MMP-9 enzymatic activities were assayed by gelatin = zymography=20 with some modifications. Culture supernatants were diluted 4:1 = with sample=20 4=C3=97 buffer (17.4% sodium dodecyl sulphate, 7% sucrose, and = phenol red in=20 Tris-HCl, pH 6.8). Sixteen microliters of sample was added to the = wells=20 under non-reducing and non-denaturing conditions. After = electrophoresis,=20 the gel was washed twice with washing buffer, followed by a brief = rinsing=20 in washing buffer with Triton X-100. The gel was then placed in = PBS, pH=20 7.4 containing 0.9 mmol CaCl₂and MgCl₂and=20 incubated overnight at room temperature on a moving platform. The = next=20 morning, the gel was stained with Coomassie Brilliant Blue G250 = (0.1% in=20 25% methanol and 10% acetic acid in water) and destained in the = same=20 solution in absence of the dye.

Statistical analyses

Data are described as the mean =C2=B1 standard deviation. For = Figure 1, = each=20 treatment was compared to its relative control using a Student = t=20 test. For Figures 2, 3, 4, = and 5,=20 treatment effects were compared using ANOVA with Bonferroni = correction.=20 The latter analysis does not apply to Figure 6, = but=20 was used to analyze the data presented in Figure 7. = The=20 data were analyzed using the SAS statistical software package = "SigmaPlot",=20 version 9.0 (SAS Institute Inc., Cary, North Carolina). The = significance=20 level was established at P < 0.05. The quantitative data were = presented=20 as three repeats from one independent experiment. This is = representative=20 of two independent experiments with similar results.

Figure 1. Evaluation of = the cell=20 viability of C6 glioma and human U-87 cells treated with berberine = or=20 As₂O₃, and co-treated with = As₂O₃₌₂₀and berberine for 24 h. C6 glioma cells (A) and = human U-87=20 cells (B) were incubated with 1, 2, 5, 10, and 20 =CE=BCM berberine or As₂O_{3 =}for 24 h=20 and the proportion of surviving cells was determined using the MTT = assay=20 as described in Methods. (C) C6 glioma and human U-87 cells were=20 co-treated with 5 =CE=BCM = As₂O₃₌₂₀and 1, 2, 5, 10, and 20 =CE=BCM = berberine for=20 24 h, and the cell growth was determined as described in Methods. = Control=20 cells were treated with As₂O₃alone. The = experiments=20 were performed in triplicate and data are presented as means = =C2=B1 SD. * and=20 ** indicate means that are significantly different when compared = to the=20 control group of C6 with P < 0.05 and P < = 0.01,=20 respectively. ^#and ^##indicate means that = are=20 significantly different when compared to the control group of U-87 = with=20 P < 0.05 and P < 0.01, = respectively.

Figure 2. Determination = of the=20 effects of berberine-As₂O₃co-treatment on = cell=20 migration and growth using the scratch-wound assay. C6 = glioma=20 cells were incubated with 5 =CE=BCM=20 As₂O₃or 10 =CE=BCM berberine=20 alone and with 5 =CE=BCM = As₂O₃₌₂₀and 10 =CE=BCM berberine for 4, = 12, and 24 h=20 and the migration was visualized as described in Methods (A). The=20 percentage of surface area filled by the C6 cells was subsequently = quantified by densitometric analyses relative to that of the = control which=20 was set at 100% as shown in the graph (B). Data are presented as = means =C2=B1=20 SD based on three independent experiments. * and ** indicate means = that=20 are significantly different when compared to the control group of = 12 h=20 incubation with P < 0.05 and P < 0.01,=20 respectively. ^#and ^##indicate means that = are=20 significantly different when compared to the control group of 24 h = incubation with P < 0.05 and P < 0.01,=20 respectively.

Figure 3. Effect of = berberine on=20 As₂O₃-inhibited motility of C6 glioma and = U-87=20 cells. (A) Cells were incubated with 5 =CE=BCM As₂O₃or 10 = =CE=BCM berberine and co-treated with 5 = =CE=BCM and 10 =CE=BCM berberine for=20 24 h. The lower and upper chemotaxis cells were separated by a=20 polycarbonate membrane. Microscopy images detected cells that = migrated=20 into the inner membrane. Magnification: =C3=97200. The cell = migration was=20 quantified by counting the number of cells that migrated into the = inner=20 membrane (B). Control cells remained untreated. The experiments = were=20 performed in triplicate and data are presented as means =C2=B1 SD. = * and **=20 indicate means that are significantly different when compared to = the=20 control group of C6 with P < 0.05 and P < = 0.01,=20 respectively. ^#and ^##indicate means that = are=20 significantly different when compared to the control group of U-87 = with=20 P < 0.05 and P < 0.01, = respectively.

Figure 4. Effect of = berberine on=20 As₂O₃-inhibited invasiveness of C6 glioma = and U-87=20 cells. (A) Cells were incubated with 5=CE=BCM As₂O₃or 10 = =CE=BCM berberine and co-treated with 5 = =CE=BCM and 10 =CE=BCM berberine for=20 24 h. Invasion through a layer of Matrigel was determined by a = Boyden=20 Chamber method as described in Methods. The invasiveness was = quantified=20 and is presented in the graph (B). Control cells remained = untreated. The=20 experiments were performed in triplicate and data are presented as = means =C2=B1=20 SD. * and ** indicate means that are significantly different when = compared=20 to the control group of C6 with P < 0.05 and P = <=20 0.01, respectively. ^#and ^##indicate means = that are=20 significantly different when compared to the control group of U-87 = with=20 P < 0.05 and P < 0.01, = respectively.

Figure 5. Actin = rearrangements in=20 C6 glioma cells treated with As₂O₃or=20 berberine. C6 glioma cells were incubated with 5 =CE=BCM As₂O₃or 10 = =CE=BCM berberine and co-treated with 5 = =CE=BCM and 10 =CE=BCM berberine for=20 24 h. Actin rearrangements were visualized by immunolocalization = using=20 anti-F-actin antibodies as described in Methods. Co-treatment with = berberine and As₂O₃resulted in actin=20 (arrows) impolarization at the edges of the cell. Actin = ruffing=20 at the edges of the elongated cells was also observed and = indicative of=20 increased migration.

Figure 6. Effect of = berberine and=20 As₂O₃on the translocation of PKC=CE=B1 and PKC=CE=B5. C6 glioma = cells were incubated=20 for 12 and 24 h with 10 =CE=BCM = berberine (Ber) or 5=20 =CE=BCM As₂O_{3 =}(As), and with 10=20 =CE=BCM berberine and 5 =CE=BCM=20 As₂O₃(A+Ber) (A, B). Cytosolic (B) and = membrane=20 fractions (A) were evaluated for the presence of PKC=CE=B1 and PKC=CE=B5 by Western blotting. = Control cells were not=20 treated in the presence of 10% Fetal Calf Serum (FCS) (N10). Cells = that=20 were not exposed to agents were used as negative control in the = presence=20 of 1% FCS (N1). The PKC levels in the respective fractions were = quantified=20 and normalized taking the =CE=B2-actin = value as a=20 loading control as presented in the graph. Each value is the = relative=20 ratio of PKCs membrane to cytosol fraction (presumably, the ratio = of=20 untreated control is 1). In addition, the nuclear protein = fractions were=20 evaluated for the presence of myc and c-jun, two transcription = factors=20 that act downstream of PKC, by Western blotting (C). The = quantitative data=20 were presented as three repeats from one independent experiment = and=20 indications are in panel A. The intensity of the protein bands was = quantified and the level relative to the N10 control band was = presented=20 (fold).

Figure 7. Berberine and=20 As₂O₃inhibit the phosphorylation of ERK and = decrease the ornithine decarboxylase (ODC), MT1-MMP, and MMP2 = protein=20 levels. C6 glioma cells were incubated for 24 h with 10 = =CE=BCM berberine (Ber) or 5 =CE=BCM=20 As₂O₃(As), and with 10 =CE=BCM=20 berberine and 5 =CE=BCM = As₂O₃₌₂₀(A+Ber). Total cell lysates were prepared and subjected to = Western=20 blot analysis. Protein levels of phosphorylated ERK1/ERK2 and=20 non-phosphorylated ERK1/ERK2, and ODC, MT1-MMP and MMP2 were = detected=20 using the respective monoclonal antibodies (A). The condition = serum free=20 media were collected for gelatin zymography analysis from C6 = glioma and=20 U-87 cells. Determined activities of these proteins were = subsequently=20 quantified by densitometric analysis with the value of controls = set at=20 100% as shown in the graph. The quantitative data were presented = as the=20 mean of three repeats from one independent experiment. Other data = in this=20 figure is presented as mean =C2=B1 SD of three independent = experiments. * and=20 ** indicate means that are significantly different when compared = to the=20 control group of C6 with P < 0.05 and P < = 0.01,=20 respectively. ^#and ^##indicate means that = are=20 significantly different when compared to the control group of U-87 = with=20 P < 0.05 and P < 0.01,=20 respectively.

Results

As₂O₃and berberine inhibit the = proliferation of=20 C6 and U-87 glioma cells

In the first instance, the effect of As₂O_{3 =}and=20 berberine, applied separately or in combination, on the viability = of C6=20 rat glioma cells and U-87 human malignant glioma cells was = investigated.=20 The cell proliferation was determined using the MTT assay = performed with=20 logarithmically growing glioma cells treated with = As₂O₃₌₂₀and berberine (Figures 1A = and 1B).=20 Within 24 h of the addition of 5 =CE=BCM=20 As₂O₃and 10 =CE=BCM berberine=20 alone on the C6 cells, proliferation was reduced to 78% and 88 %,=20 respectively (P < 0.05). Treatment with the same=20 concentrations of As₂O₃or berberine did not = elicit=20 marked cytotoxic effects on U-87 cells (Figure 1B).=20 However, at a concentration of 20 =CE=BCM, decreased=20 cell viability was observed. Interestingly, based on the trypan = blue dye=20 exclusion assay, it was observed that 5 =CE=BCM=20 As₂O₃and 10 =CE=BCM berberine=20 combined treatment exhibited a minor effect on the viability of C6 = and=20 U-87 cells (data not shown). A dose-dependent effect on the = inhibition of=20 the C6 or U-87 glioma cell proliferation at lower agent = concentrations was=20 observed (Figure 1C).=20 Co-treatment with 5 =CE=BCM = As₂O₃₌₂₀and 10 =CE=BCM berberine, = concentrations used=20 for all further analyses, led to an inhibition of the cell = proliferation=20 and indicated no major signs of apoptosis, suggesting that another = biological or pharmacological function played a role.

Berberine enhances As₂O₃-mediated = reduction in=20 glioma cell migration and invasion

Using the scratch-wound assay, a continuous rapid movement was = observed=20 for all cells, but a resultant movement of a glioma cell migration = front=20 was clearly evident at 24 h, where a highly confluent = (90%=E2=80=93100%) monolayer=20 region gradually migrated into the cell-free 'scratch' region = (Figure 2A). = In=20 the presence of 5 =CE=BCM=20 As₂O₃, migration was significantly reduced = after 12=20 and 24 h of incubation, whereas co-treatment with 5 =CE=BCM As₂O₃and 10 = =CE=BCM berberine led to a virtually = complete inhibition of=20 cell migration (Figures 2A = and 2B).

Cell proliferation and invasive behaviors are important = characteristics=20 of cancer cells and indicators of malignance, and both are targets = of=20 anti-cancer agent development [44]. = The Boyden=20 chamber assay was used to evaluate the inhibitory effect of = combined=20 treatment with As₂O₃and berberine on C6 or = U-87=20 glioma cell migration and invasiveness. The observations revealed = that=20 berberine-As₂O₃co-treatment resulted in a=20 remarkable inhibition of glioma cell migration as compared to = either=20 treatment alone (Figure 3), = which=20 supported the results obtained with the scratch-wound assay = (Figure 2). = In=20 addition, co-treatment with As₂O₃and = berberine also=20 exhibited a significant anti-invasive effect on glioma cells = (Figure 4), = which=20 enhanced the dose-response relationship.

An important characteristic of advanced cancer development = metastasis,=20 during which cancer cells migrate to other tissues and organs, is = the=20 rearrangement of the cytoskeleton of migrating cells [45]. = The=20 cytoskeleton changes in C6 glioma cells treated with 5 =CE=BCM As₂O₃or 10 = =CE=BCM berberine, and co-treated with=20 As₂O₃and berberine, were visualized using = F-actin=20 specific antibodies that bind to F-actin (filopodia; yellow arrow = head,=20 Figure 5), = and=20 revealed structural rearrangements in the actin cytoskeleton that = were=20 observed upon cell spreading and ruffling (Figure 5).

Berberine prevents cytosol-to-membrane translocation of = PKC=CE=B1 and PKC=CE=B5 isoenzymes

Recent studies report that overexpressed or hyperactive PKC is = among=20 the most distinguishing characteristics of central nervous system = tumors=20 [46]. = The=20 PKC=CE=B1 and PKC=CE=B5 activity = levels seem to be=20 increased in malignant gliomas and mechanisms related to tumor = cell=20 invasion, and metastasis is activated [47,48]. = To further=20 evaluate the effect of chemotherapeutic agents such as=20 As₂O₃in the PKC signaling pathway, we = examined=20 whether berberine enhanced As₂O₃-mediated = modulation=20 of the translocation of PKC=CE=B1 and = PKC=CE=B5 isoenzymes=20 using Western blot analyses. Remarkably, 10 =CE=BCM=20 berberine had a dramatic effect on the cytosol-to-membrane = translocation=20 of PKC=CE=B1 and PKC=CE=B5 after 12 = and 24 h incubation=20 (Figures 6A = and 6B).=20 Furthermore, the combined treatment with As₂O_{3 =}and=20 berberine resulted in inhibition of the activation of PKC=CE=B1 and PKC=CE=B5, but not PKC=CE=B4=20 (Figures 6A = and 6B), = as=20 compared to the negative control (1% fetal calf serum). Once = activated,=20 PKC can transmit signals to the nucleus via MAPK-mediated cascades = and=20 activated ERKs can induce the production of transcription factors=20 including myc and jun [49,50]. = To further=20 investigate the effect of As₂O₃-berberine=20 co-treatment on PKC-mediated signaling, the nuclear levels of = those=20 transcription factors were determined. Figure 6C=20 illustrates that the levels of myc and jun in nuclei were = significantly=20 reduced after treatment with 10 =CE=BCM berberine in=20 the presence of 5 =CE=BCM = As₂O₃₌₂₀for 24 h.

Inhibitory effect of berberine and A₂O_{3 =}on the=20 activation of MMP-2 associated with invasiveness of glioma cells = via=20 interference with PKCs

Activated PKCs enable the expression of genes encoding enzymes = that are=20 involved in cell proliferation and invasion [30,46,50] = such as the=20 gene encoding ornithine decarboxylase (ODC), an enzyme that plays = a role=20 in cell transformation and excessive extracellular matrix = degradation [51]. = Because=20 the observations presented above strongly suggest that berberine = and=20 As₂O₃act via the inhibition of PKC = signaling, the=20 activation of three PKC-dependent key factors was examined by = Western blot=20 analyses. These experiments demonstrated that the=20 berberine-As₂O₃-mediated enhanced = suppression of ERK=20 phosphorylation led to a decrease of the levels of ODC, MT1-MMP, = and MMP-2=20 in C6 glioma cells to values that resembled those observed in = negative=20 controls (Figure 7A).

Extracellular matrix breakdown is pivotal for cellular = invasion,=20 indicating that matrix-degrading proteinases are essential for = tumor cell=20 metastasis [28]. = Therefore,=20 we determined the activity of MMP-2 by gelatin zymography after 24 = h=20 incubation with 5 =CE=BCM = As₂O₃₌₂₀and 10 =CE=BCM berberine in C6 = or U-87 glioma=20 cells. A significant decrease in the activity of MMP-2 was = observed after=20 24 h exposure to As₂O₃and berberine (Figure = 7B),=20 likely explaining why co-treatment with berberine and=20 As₂O₃strongly affects glioma cell migration = and=20 invasiveness.

Discussion

Berberine is a naturally occurring alkaloid that exhibits = various=20 pharmacological effects. Clinical trials and studies in animal = model=20 systems have demonstrated that berberine has anti-microbial, = vulnerary,=20 cardiovascular [52],=20 immunostimulatory, anti-hemorrhagic, and anti-inflammatory = properties, as=20 well as selective repression of the growth of several carcinoma = cells=20 without causing cytotoxicity in the liver [32,53]. = Control=20 experiments using rat primary cortical astrocytes illustrated that = treatment with 5 =CE=BCM = As₂O₃₌₂₀or 20 =CE=BCM berberine, or = co-treatment with 5=20 =CE=BCM As₂O_{3 =}and 20 =CE=BCM berberine, did not cause any = detectable toxic=20 effects (data not shown). However, the effect of berberine on = migration=20 and invasion of glioma cells at relatively low concentrations was=20 striking, particularly in the presence of = As₂O₃.=20 Most interesting was the enhanced effect that=20 As₂O₃-berberine co-treatment had on glioma = cell=20 migration and invasiveness, as 10 =CE=BCM berberine=20 significantly lowered the concentration of As₂O₃₌₂₀required to obtain an antiproliferation effect on human = glioma=20 cells. This dose-lowering effect, unique to this study, could not = be=20 exploited in other studies in which tumor cells were treated with = either=20 berberine [37,38] = or=20 As₂O₃[14].

As₂O₃has considerable efficacy in the = treatment=20 of relapsed APL, inducing partial differentiation and promoting = apoptosis=20 of malignant promyelocytes [54,55]. = The=20 mechanisms underlying As₂O₃-mediated = apoptosis are=20 only beginning to be understood, but appear to be distinct from = those=20 employed by traditional cytotoxic agents. The intracellular = glutathione=20 redox system represents the best-characterized mechanism of=20 As₂O₃sensitivity [39,55]. = These=20 findings underscore the importance of understanding how = differences in=20 cell types or cellular environments might affect the action of=20 As₂O₃. A determination of the factors that = mediate=20 sensitivity to As₂O₃will allow the use of = this=20 agent in such a way as to optimize therapeutic outcomes and = minimize=20 toxicity in the treatment of various malignancies. To date, = evidence that=20 the development of resistance to chemotherapy affects the response = to=20 arsenic is scarce. Thus, As₂O₃represents a = novel=20 chemotherapeutic agent worth for continuous investigation, = particularly=20 when used in combination with another agent such as berberine that = exhibits an enhanced inhibitory effect, which allows lowering the = working=20 As₂O₃concentration.

In previous studies, cell movement through tissue has been = observed to=20 play a primary role in cancer progression. This process requires a = series=20 of distinct, but concerted, biological events in which the actin=20 cytoskeleton plays an essential role. These events include tumor = cell=20 attachment to extracellular matrix (ECM) components, and the = degradation=20 of the matrix by tumor cell-associated proteases [28,56]. = It has=20 long been known that in most cell types, one or more PKC isoforms=20 influence the morphology of the F-actin cytoskeleton and = overexpressed or=20 hyperactive PKC is among the most distinguished characteristics of = malignant central nervous system tumors including C6 or U-87 = glioma cells=20 [46,57]. = In certain=20 tumors, a reduction in PKC=CE=B1 and = PKC=CE=B5 expression=20 merely results in growth inhibition, so that a cytotoxic effect = requires=20 induction of apoptosis through activation of PKC=CE=B4 [58]. = Exposure=20 of C6 cells in the presence of As₂O₃to = berberine=20 exhibits a significant effect characterized by alterations in cell = shape=20 and actin cytoskeleton changes. Moreover, the data presented = herein=20 suggests that the cytosol-to-membrane translocation of PKC=CE=B1 and PKC=CE=B5 is slightly suppressed = by=20 As₂O₃, and this inhibition is enhanced upon=20 co-treatment with berberine for 24 h, although berberine itself = did cause=20 inactivation of PKC=CE=B1 and = PKC=CE=B5 but not PKC=CE=B4. Similar to the action of the PKC = signaling pathway,=20 the expression on myc, c-fos, ODC, MT1-MMP, and MMP-2 as well as = the=20 phosphorylation of ERK by co-treatment could be abolished.

Arsenic-containing compounds have been used for the treatment = of cancer=20 for hundreds of years in both traditional Chinese and Western = medicine [59]. = Only a few=20 reports are available that describe the underlying mechanisms of = the=20 action of specific chemotherapeutic agents used to treat cancer=20 metastasis. In this study, we demonstrate for the first time that=20 berberine sensitizes malignant glioma cells for=20 As₂O₃-mediated suppression of migration and=20 invasiveness likely via decreased PKC signaling and MMP-2 = activation in=20 the extracellular matrix. The involvement of PKC signaling is not = yet=20 undoubtedly proven, as the effect of As₂O_{3 =}and=20 berberine on glioma tumor cells overexpressing PKC=CE=B1 and PKC=CE=B5 remains to be = investigated. Furthermore, we=20 tried to investigate the dual effect that berberine has on glioma = cells.=20 First, berberine exhibits an anti-oxidant activity thereby = protecting=20 healthy cells against the As₂O₃-induced = production=20 of toxic reactive oxygen species [5,33,53]. = Second,=20 berberine may reduce the As₂O₃-mediated = carcinogenic=20 effect by inhibiting PKC=CE=B1 = translocation in the=20 mouse epidermal JB6 cells [60]. = This dual=20 effect is particularly important in a co-treatment with=20 As₂O₃because As₂O₃is = a=20 carcinogen [10]; = however,=20 upon co-treatment with berberine, the working concentration of=20 As₂O₃can be significantly lowered, while, = in=20 addition, healthy cells are protected against possible adverse = side=20 effects caused by exposure to As₂O₃.

Invasion and metastasis inhibitors are effective (alone or in=20 combination therapy with other agents) in restraining new tumor = formation=20 when earlier therapy or surgery has failed. Therefore, it appears = that a=20 combination of As₂O₃and berberine inhibits = the=20 proliferation and invasion of glioma cells. Although future = studies are=20 required to experimentally link the various pathways that appear = to be=20 involved in the berberine/As₂O₃-mediated = suppression=20 of glioma cell migration and invasiveness, to better understand = the=20 molecular mechanisms that occur in the extracellular matrix = surrounding=20 glioma cells during tumor metastasis, to investigate novel methods = to=20 specifically deliver, for instance, berberine and = As₂O₃₌₂₀to glioma tumor cells, and to investigate the effectiveness = and=20 safeness of berberin-As₂O₃co-treatment in a = clinical setting, this study forms the basis for the design of = novel=20 therapeutic strategies to ameliorate and prevent human glioma = formation.=20 Furthermore, the observations presented herein suggest that (i) = berberine=20 should be further considered as a complementary antitumor = treatment in=20 clinical applications, and (ii) application of berberine has a = dual=20 ameliorating effect as it enhances the antitumor effect of=20 As₂O₃while it allows a lower working = concentration=20 of As₂O₃by which its global carcinogenic = effect to=20 healthy cells, mediated by the fact that elemental arsenic can = enter the=20 cerebrospinal fluid [61], = is=20 diminished.

Conclusion

We demonstrate that berberine enhances inhibition of the = migration and=20 invasiveness of glioma cells mediated by = As₂O₃, or,=20 in other words, that a concentration as low as 10 =CE=BCM of the natural compound berberine is = sufficient to=20 significantly lower the As₂O₃concentration = needed=20 to obtain its antiproliferation effect in a rat and human = malignant glioma=20 cell line. While not yet undoubtedly proven, our results suggest = that this=20 effect is likely mediated by the disturbance in PKC signaling = which leads=20 to a reduction of the level of MMP-2, a metalloprotease involved = in cancer=20 cell migration during metastasis.

List of abbreviations

APL, acute promyelocytic leukemia; ECM, extracellular matrix; = ERK,=20 extracellular signal-regulated kinase; MAPK, mitogen-activated = protein=20 kinase; MMP, matrix metalloproteinase; MT1-MMP, membrane type-1 = matrix=20 metalloproteinase; ODC, ornithine decarboxylase; PBS, = phosphate-buffered=20 saline; PKC, protein kinase C

Competing interests

The author(s) declare that they have no competing = interests.

Authors' contributions

LTH conceived, designed, and performed the study, and prepared = the=20 manuscript. KHC performed data analysis, and provided cell lines = and=20 technical assistance to LTH. Chou FP supervised work and = manuscript=20 review. LFJ, supervised the entire study and manuscript = revision.

Acknowledgements

The study was supported by a grant of the Taichung Veterans = General=20 Hospital (TCVGH-953701A).

The author would like to thank Dr. Chun-Jung Chen for helping = to=20 perform controlexperiments using rat primary cortical = astrocytes.

^=E2=80=A0These authors contributed equally to this = work.

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Pre-publication history

The pre-publication history for this paper can be accessed = here:

http://www.bi= omedcentral.com/1471-2407/8/58/prepub

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Berberine enhances inhibition of glioma tumor cell migration = and=20 invasiveness mediated by arsenic trioxide

Abstract

Background

Methods

Results

Conclusion

Background

Methods

Cell culture

Chemical reagents and antibodies

Cell growth and proliferation assay

Boyden chamber assay

Matrigel invasion assay

Scratch assays

Preparation of total cell extracts and immunoblot = analysis

Immunofluorescent staining assay

PKC translocation assay

Zymography

Statistical analyses

Results

As2O3 and berberine inhibit the = proliferation of=20 C6 and U-87 glioma cells

Berberine enhances As2O3-mediated = reduction in=20 glioma cell migration and invasion

Berberine prevents cytosol-to-membrane translocation of = PKC=CE=B1 and PKC=CE=B5 isoenzymes

Inhibitory effect of berberine and A2O3 = on the=20 activation of MMP-2 associated with invasiveness of glioma cells = via=20 interference with PKCs

Discussion

Conclusion

List of abbreviations

Competing interests

Authors' contributions

Acknowledgements

References

Pre-publication history

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As₂O₃and berberine inhibit the = proliferation of=20 C6 and U-87 glioma cells

Berberine enhances As₂O₃-mediated = reduction in=20 glioma cell migration and invasion

Inhibitory effect of berberine and A₂O_{3 =}on the=20 activation of MMP-2 associated with invasiveness of glioma cells = via=20 interference with PKCs