Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Berberine is an isoquinoline derivative alkaloid isolated
from many medicinal herbs, such as Rhizoma coptidis
and Cortex phellodendri. It is widely used in traditional
Chinese medicine for antimicrobial and anti-inflammatory
activities. In recent years, berberine has been reported to
have a wide range of pharmacological effects, including
immunological regulation[1], myocardial
protection[2], inhibition of tumor cell
proliferation[3], and
invasion[4]. Recently, we reported the inhibitory effect of berberine on the invasion
and migration of lung carcinoma
cells[5].
Cyclin D1 is a member of the G1 cyclin family involved in
the regulation of the G1/S transition of the cell
cycle[6]. The cyclin D1/cyclin-dependent kinase 4 (CDK4) complex can
hyperphosphorylate retinoblastoma tumor suppressor
protein 1 (Rb1)[7], leading to the dissociation of E2
promoter-binding protein dimerization partners (E2F) from the Rb1/E2F
complex[8]. Dissociated E2F induces the transcription of cyclin E
and other genes required for entry into the S phase. Cyclin D1
is frequently overexpressed in a wide range of cancers. The
nuclear accumulation of cyclin D1 induces uncontrolled
proliferation in normal human cells, which may facilitate the
development of invasive cancer[9]. The cyclin D1 expression is
under complex regulation and is markedly influenced by the
activating protein-1 (AP-1), NF-κB, and β-catenin/T cell
factor (TCF) signaling pathways[10-12]. A number of compounds
targeting these signaling pathways can indirectly attenuate
the cyclin D1 expression to mediate cell cycle arrest.
AP-1 is a sequence-specific transcription factor
composed of homodimers or heterodimers of the Jun family
(c-Jun, Jun D, and Jun B) or heterodimers of the Jun family
members with any of the Fos family members (c-Fos, Fos B,
Fra1, and Fra2). AP-1 has long been associated with
proliferation. AP-1 directs the expression of a critical target
gene or genes, such as the cyclin D1 gene, in response to
cytokines, stress, and mitogenic
signals[13]. The promoter for
CCND1 (encoding cyclin D1) contains an AP-1 motif,
and the ectopic expression of c-Jun induces the cyclin D1
mRNA expression[14].
The present study was performed to verify the
suppressive effect of berberine on the proliferation of the human
pulmonary giant cell carcinoma cell line PG and demonstrate
the mechanisms behind the antitumoral effects of berberine.
Materials and methods
Cell culture Human pulmonary giant cell carcinoma cell
line PG (Peking University Medical Center, Beijing,
China)[15] were cultivated in RPMI-1640 medium supplemented with
10% fetal bovine serum. Cell cultures were maintained in a
37 °C incubator under a humidified 5%
CO2 atmosphere, and were routinely subcultured by trypsinization. All experiments
were performed on logarithmically-growing cells.
Cell proliferation/viability assay The PG cells were seeded
onto 96-well culture plates and incubated for 24 h at 37 °C and
then treated with berberine (10, 20, and 40 µg/mL;
NICPBP, Beijing, China) or without. After 24 or 48 h, cell proliferation
was assayed by using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Promega, Madison, WI,
USA). The absorbance was recorded with a microplate reader
(model 550, Bio-Rad, Hercules, CA, USA); viability was
determined as
(%)=(A570_A630)
sample/(A570_A630
) control×100%.
Cell cycle analysis The PG cells were treated with
berberine (0, 20, and 40 µg/mL) for 48 h. The cells were then
harvested, washed with cold phosphate-buffered saline
(PBS), and processed for cell cycle analysis. Briefly,
2×105 cells were resuspended in 0.5 mL cold PBS, to which cold
ethanol (70%, 5 mL) was added; the cells were then
incubated for 1 h at 4 °C. After centrifugation, the pellet was
washed with cold PBS, suspended in 0.3 mL PBS, and
incubated with 50 µL RNase (1 mg/mL) for 30 min at 37 °C. The
cells were kept on ice for 10 min and incubated with 500
µL propidium iodide (50 mg/L) for 30 min in the dark. The cell
cycle distribution of the cells of each sample was then
determined by using a FACSCalibur instrument (BD Biosciences,
San Jose, CA, USA) equipped with FACSort Cell Quest software.
RT_PCR After treatment with or without berberine, total
RNA was extracted. Three micrograms of total RNA was
reverse transcribed using SuperScript III reverse transcriptase
(Invitrogen, Carlsbad, CA, USA) at 50 °C for 2 h. The 20 µL
PCR reaction contained 2 µL of 10× Taq
buffer, 0.5 µL of each primer 10 µmol/L; cyclin D1: 5´-GCG AGG AAC AGA AGT
GCG-3´ [sense] and 5´-GAA GCG TGT GAG GCG GTA-3´
[antisense], and GAPDH: 5´-GGG GAA GGT GAA GGT
CGG-3´ [sense] and 5´-ATG AGT CCT TCC ACG ATA CCA A-3´
[antisense], 0.2 µL Taq DNA polymerase (Tiangen, Beijing,
China), 0.5 µL of 10 mmol/L dNTP, and 1 µL cDNA. GAPDH
was used as an internal loading control. The expected sizes
of the PCR products for cyclin D1 and GAPDH were 494 and
522 bp, respectively.
Cell transfection and luciferase assay The PG cells were
seeded onto 96-well plates and incubated for 24 h at 37 °C.
For each well, 100 ng pAP-1-Luc (or pNF-κB-Luc) and 10 ng
pRL-TK (Promega, USA) were mixed and cotransfected
using the calcium phosphate precipitation method according
to standard protocols[16].
The cells were treated with the indicated concentrations
of berberine for 6 h after transfection, and incubated for an
additional 24 h. The stimulus group was treated with phorbol
myristate acetate (PMA and ionomycin (P+I) for 6 h before
being lysed. The cells were then lysed in 40
µL of passive lysis buffer (Promega, USA). Firefly luciferase and Renilla
luciferase activities were measured with 10 µL cell lysate
using the Dual luciferase reporter assay system (Promega,
USA) in a GENios pro reader (Tecan, Hombrechtikon,
Switzerland). Relative activity was defined as the ratio of
firefly luciferase activity to Renilla luciferase activity and
was calculated by dividing the luminescence intensity
obtained in the assay for firefly luciferase by that obtained for
Renilla luciferase.
Western blot analysis After the berberine treatments,
the PG cells were harvested, and whole-cell lysates were
prepared. Equal amounts of protein samples were separated
by SDS_PAGE gel and blotted onto nitrocellulose (NC)
membrane (Millipore, Bedford, MA, USA). After blocking, the
membranes were incubated at 4 °C with antibodies against
c-Jun (1:500) or cyclin D1 (1:200; Santa Cruz Biotechnologies,
Santa Cruz, CA, USA). β-Actin was used as an internal
loading control. The blots were then washed and incubated for
1 h with horseradish peroxidase-labeled secondary antibody
(1:4000; Zhongshan Golden Bridge, Beijing, China).
Immunoreactive bands were visualized with a SuperSignal West
Pico chemiluminescnet kit (Pierce, Rockford, IL, USA).
Electrophoretic mobility shift assay Nuclear extracts were
prepared by using a NE-PER nuclear and cytoplasmic
extraction reagent kit (Pierce, USA) according to standard
protocols. The CCND1 AP-1 site, the wild-type collagenase AP-1 site,
and a mutant CCND1 AP-1 site were synthesized as
complementary oligodeoxyribonucleotide strands. The sequence of
the CCND1 promoter AP-1 site
oligodeoxyribonucleotides was 5´-TCC ATT CTG ACT CAT TTT TTT TAA-3´, and the
sequence of the mutant AP-1 site was 5´-TCC ATT CTG cCg
CAT TTT TTT TAA-3´. The sequence of the wild-type
collagenase AP-1 oligodeoxyribonucleotides was 5´-CGC TTG
ATG AGT CAG CCG GAA-3´[17].
The DNA binding ability of AP-1 in the nuclear extracts was
assessed by electrophoretic mobility shift assay
(EMSA)[18] with biotin-labeled, double-stranded, wild-type collagenase
AP-1 oligonucleotides and CCND1 AP-1 oligonucleotides.
EMSA was carried out by using the Lightshift
chemiluminescent EMSA kit (Pierce, USA). Specific binding was
confirmed by using a 250-fold excess of an unlabeled probe as a
specific competitor. Protein_DNA complexes were separated
by using a 6% non-denaturing acrylamide gel
electrophoresis and then transferred to positively-charged nylon
membranes and cross-linked by UV irradiation. Gel shifts were
visualized with streptavidin horseradish peroxidase
according to standard protocols.
Statistical analysis All data are expressed as
mean±SD. Student's unpaired t-test was used to compare differences
between 2 groups. Figures were obtained from at least 3
independent experiments with similar patterns.
Results
Berberine inhibits the proliferation and viability of PG
cells The PG cells were treated with 0, 10, 20, and 40 µg/mL
berberine for 24 and 48 h. The treatment of the PG cells with
berberine (10_40 µg/mL) resulted in a significant reduction
in cell proliferation/viability as assessed by MTT assay,
ranging from 27% to 36% (P<0.01) after 24 h, and 63% to
73% (P<0.01) after 48 h (Figure 1A).
Berberine induces G1 phase cell cycle arrest in PG cells
As we found a significant growth inhibitory effect of
berberine on PG cells, we determined the possible inhibitory
effect of berberine on cell cycle progression. The treatment
of PG cells with berberine (20 and 40 µg/mL) for 48 h resulted
in a significantly higher number of cells in the
G1 phase at the concentrations used: 20 µg/mL
(47%, P<0.01) and 40 µg/mL (53%,
P<0.01), compared with the non-berberine-treated
control (36%; Figure 1B). In each case, there was a concomitant
reduction in the number of cells in the S and
G2_M phases. These data suggested that the inhibition of cell proliferation
in PG cells by berberine may be associated with the
induction of G1 arrest.
Berberine suppresses the cyclin D1 expression
Based on the preliminary assays in which we determined the
effects of berberine on cell proliferation and viability, in order
to minimize the cytotoxic effect of berberine, we selected
doses lower than 10 µg/mL for further mechanism studies.
Cyclin D1 is a downstream molecule regulated by the
AP-1 and NF-κB signaling pathways, and is a key molecule that
controls the cell cycle entry from the
G1 phase to the S phase. We applied RT_PCR and immunoblotting to check the mRNA
and protein expressions of cyclin D1. Treatment with
berberine at different concentrations significantly downregulated
the cyclin D1 mRNA and protein expressions (Figure 2).
Berberine inhibits AP-1 transcriptional activity
Luciferase activity in the cells with the AP-1 construct was
significantly reduced by treatment with berberine at 2.5, 5,
and 10 µg/mL, whereas luciferase activity in the cells
containing the NF-κB construct showed no statistically
significant changes in the presence of berberine (Figure 3A, 3B).
After treatment with PMA (60 ng/mL) and ionomycin (1.25
µmol/L), the cells with the AP-1 construct showed a
significant decrease in luciferase activity in the presence of
berberine (Figure 3C). The results demonstrated that berberine
could suppress the AP-1 pathway in PG cells, but had no
significant effect on the NF-κB pathway, Moreover,
berberine significantly inhibited the AP-1 pathway when it was
activated by the stimulus.
Berberine inhibits the c-Jun expression The luciferase
assay results suggest that berberine inhibits the activity of
the AP-1 pathway. c-Jun is primarily a positive regulator of
cell proliferation[18]. The activated c-Jun-containing AP-1
complex induces the transcription of positive regulators of
cell cycle progression, such as cyclin D1. We examined the
effects of berberine on the expression of c-Jun by
immunoblotting using the same culture and treatment conditions as before.
Berberine treatment (2.5, 5, and 10 µg/mL) significantly
decreased the expression of c-Jun (Figure 3D).
Berberine decreases transcription factors binding to
the CCND1 gene AP-1 motif To validate the previous
results further, EMSA were performed by using
oligonucleotides containing the wild-type collagenase AP-1 site as the
probe. The PG cells were incubated in the presence of
different concentrations of berberine for 24 h, and nuclear extracts
were then prepared and analyzed for AP-1 DNA
binding activity. The results indicated that the activity of AP-1
decreased dramatically when the cells were treated with
berberine (Figure 4A). These data were consistent with the
reporter gene analysis. The binding of transcription factors
to the CCND1 gene AP-1 motif was detected by using
oligonucleotides containing the CCND1 AP-1 site as the probe.
The binding decreased in the presence of berberine, while
the mute probes had no effect on the combination (Figure
4B). The results suggested that berberine blocked the cyclin
D1 expression, at least in part, by decreasing the expression
or DNA binding activity of members of the AP-1
transcription factor family.
Discussion
Berberine is one of the major components of Coptis
chinensis, which was frequently used in proprietary herbal
medicines to treat inflammation in Europe and Asia.
Berberine exhibits a broad spectrum of antimicrobial activity by
inhibiting fungal, yeast, and bacterial proliferation with no
toxicity. Studies have shown that berberine exerts a wide
range of effects on angiogenesis[19], cell proliferation,
apoptosis[20], cell
cycle[21], and tumor metastasis in
various in vivo and in vitro models. It is then valuable to investigate
the mechanisms of such antitumoral effects of berberine.
The inhibitory effect of berberine on AP-1 activity has
been reported previously[22,23]. However, the action
mechanism of berberine on AP-1 remains unknown. The
downstream target genes of AP-1, containing putative
AP-1-binding sites in the promoters, are involved in many critical
cellular functions, such as cell cycle progression and DNA
synthesis[18]. CCND1 is a prototype of such a gene that may
directly link AP-1 to cell cycle progression. Cyclin D1 antisense
treatment blocks mammary tumor growth in
vivo[24], and cyclin D1 knockout mice are resistant to mammary tumor
development by ras[25,26]. In the present study, we demonstrated
that berberine inhibits the expression of cyclin D1 by
downregulating AP-1 transcriptional activity. Thus berberine
arrests cell cycle progression and exerts antitumoral effects.
In summary, our results show that berberine significantly
suppresses AP-1 transcriptional activity, leading to the
inhibition of the cyclin D1 expression through the suppression
of the c-Jun expression and binding of transcription factors
to the CCND1 gene AP-1 motif. Berberine arrests cell cycle
progression and proliferation of PG cells. These findings
elucidate one of the important mechanisms behind the
antitumoral effects of berberine as a regulator of cyclin D1.
References
1 Xu LH, Liu Y, He XH. Inhibitory effects of berberine on the
activation and cell cycle progression of human peripheral
lymphocytes. Cell Mol Immunol 2005; 2: 295_300.
2 Zhao HP, Hong Y, Xie JD, Xie XR, Wang J, Fan JB. Effect of
berberine on left ventricular remodeling in renovascular
hypertensive rats. Yao Xue Xue Bao 2007; 42: 336_41.
3 Letasiova S, Jantova S, Miko M, Ovadekova R, Horvathova M.
Effect of berberine on proliferation, biosynthesis of
macromolecules, cell cycle and induction of intercalation with DNA, dsDNA
damage and apoptosis in Ehrlich ascites carcinoma cells. J Pharm
Pharmacol 2006; 58: 263_70.
4 Peng PL, Hsieh YS, Wang CJ, Hsu JL, Chou FP. Inhibitory effect
of berberine on the invasion of human lung cancer cells via
decreased productions of urokinase-plasminogen activator and
matrix metalloproteinase-2. Toxicol Appl Pharmacol 2006; 214:
8_15.
5 Hao Y, Xu BW, Zheng H, Wang Q, Qiu QY, Huang QF. Effect of
berberine on invasion and migration of PG cells from a high
metastatic human giant lung carcinoma cell line. Chin J
Pathophysiol 2007; 23: 474_8.
6 Matsushime H, Roussel MF, Ashmun RA, Sherr CJ.
Colony-stimulating factor 1 regulates novel cyclins during the G1 phase
of the cell cycle. Cell 1991; 65: 701_13.
7 Kato JY, Matsuoka M, Strom DK, Sherr CJ. Regulation of cyclin
D dependent kinase 4 (cdk4) by cdk4-activating kinase. Mol
Cell Biol 1994; 14: 2713_21.
8 Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct
binding of cyclin D to the retinoblastoma gene product (pRb) and
pRb phosphorylation by the cyclin D-dependent kinase CDK4.
Genes Dev 1993; 7: 331_42.
9 Gautschi O, Ratschiller D, Gugger M, Betticher DC, Heighway J.
Cyclin D1 in non-small cell lung cancer: a key driver of
malignant transformation. Lung Cancer 2007; 55: 1_14.
10 Eferl R, Wagner EF. AP-1: a double-edged sword in tumorigenesis.
Nat Rev Cancer 2003; 3: 859_68.
11 Joyce D, Bouzahzah B, Fu M, Albanese C, D'Amico M, Steer J,
et al. Integration of rac-dependent regulation of cyclin D1
transcription through a nuclear factor-κB-dependent pathway. J Biol
Chem 1999; 274: 25 245_9.
12 Natsume H, Sasaki S, Kitagawa M, Kashiwabara Y, Matsushita A,
Nakano K, et al. β-Catenin/Tcf-1-mediated transactivation of
cyclin D1 promoter is negatively regulated by thyroid hormone.
Biochem Biophys Res Commun 2003; 309: 408_13.
13 Shaulian E, Karin M. AP-1 in cell proliferation and survival.
Oncogene 2001; 20: 2390_400.
14 Fu M, Wang C, Zhang X, Pestell RG. Signal transduction
inhibitors in cellular function. Methods Mol Biol 2004; 284: 15_36.
15 Wu BQ, Sun YK, Zheng J, Fang WG, Wang JL. Metastatic
behavior of cultured human lung carcinoma cell lines in nude mice. In:
Rygaard J, Brünner N, Græm N, Spang-Thomsen M.
Immune-deficient animals in biomedical research. Proceedings of the 5th
International Workshop on Immune-deficient Animals;
Copenhagen, Denmark, Oct 13-16, 1985; Karger, Basel,
Switzerland; 20 May 1987; p 296_302.
16 Xia D, Li X, Lou Y, Han W, Ding P, Zhang Y,
et al. Overexpression of chemokine-like factor 2 promotes the proliferation and
survival of CaCl2 skeletal muscle cells. Biochim Biophys Acta 2002;
1591: 163_73.
17 Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A,
et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin
D1 promoter through distinguishable regions. J Biol Chem 1995;
270: 23 589_97.
18 Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin
J, et al. Transcriptional interference between c-Jun and the
glucocorticoid receptor: mutual inhibition of DNA binding due to
direct protein-protein interaction. Cell 1990; 62: 1205_15.
19 Lin S, Tsai SC, Lee CC, Wang BW, Liou JY, Shyu KG. Berberine
inhibits HIF-1α expression via enhanced proteolysis. Mol
Pharmacol 2004; 66: 612_9.
20 Jantova S, Cipak L, Letasiova S. Berberine induces apoptosis
through a mitochondrial/caspase pathway in human
promonocytic U937 cells. Toxicol In Vitro 2007; 21: 25_31.
21 Lin CC, Lin SY, Chung JG, Lin JP, Chen GW, Kao ST.
Down-regulation of cyclin B1 and up-regulation of wee1 by berberine
promotes entry of leukemia cells into the
G2/M-phase of the cell cycle. Anticancer Res 2006; 26: 1097_104.
22 Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H.
Inhibition of activator protein 1 activity by berberine in human
hepatoma cells. Planta Med 1999; 65: 381_3.
23 Mitani N, Murakami K, Yamaura T, Ikeda T, Saiki I. Inhibitory
effect of berberine on the mediastinal lymph node metastasis
produced by orthotopic implantation of Lewis lung carcinoma.
Cancer Lett 2001; 165: 35_42.
24 Lee RJ, Albanese C, Fu M, D'Amico M, Lin B, Watanabe G,
et al. Cyclin D1 is required for transformation by activated Neu and is
induced through an E2F-dependent signaling pathway. Mol Cell
Biol 2000; 20: 672_83.
25 Fantl V, Stamp G, Andrews A, Rosewell I, Dickson C. Mice
lacking cyclin D1 are small and show defects in eye and
mammary gland development. Genes Dev 1995; 9: 2364_72.
26 Yu Q, Geng Y, Sicinski P. Specific protection against breast
cancers by cyclin D1 ablation. Nature 2001; 411: 1017_21.
|
