Volume 103, Issue 4, April 2015, Pages 1098–1106
Original article
Objective
To
determine whether berberine (BBR), a naturally occurring plant-derived
alkaloid, inhibits the proliferation of human uterine leiomyoma (UtLM)
cells.
Design
Laboratory research.
Setting
Laboratory.
Patient(s)
UtLM and normal human uterine smooth muscle (UtSMC) cell lines.
Intervention(s)
Treatment with [1] BBR (10, 20, and 50 μM), [2] BBR (20 and 50 μM) and/or 17β-estradiol (E2; 10 and 100 nM), and [3] BBR (20 and 50 μM) and/or progesterone (P4; 10 and 100 nM) for 24 or 72 hours.
Main Outcome Measure(s)
Cell proliferation, cell cycle, apoptosis, and related genes expression were determined.
Result(s)
BBR
inhibited UtLM cell proliferation by inducing G2/M cell cycle arrest
and apoptosis. Cell cycle G2/M phase-related genes were altered by BBR
treatment: the expression of cyclin A1, cyclin B1, and Cdk1 were
down-regulated, while Cdk4, p21, and p53 were up-regulated. BBR-treated
cells stained positively for annexin V and manifested increased BAX
expression. E2- and P4-induced UtLM cell
proliferation was blocked by BBR treatment. In marked contrast, even the
highest concentration of BBR (50 μM) did not influence cell
proliferation in UtSMC cells.
Conclusion(s)
BBR selectively inhibits cellular proliferation and blocks E2- and P4-induced
cell proliferation in UtLM but not in normal UtSMC cells. In addition,
BBR did not demonstrate cytotoxicity effects in normal human UtSMCs. Our
results suggest BBR could be a potential therapeutic agent for the
treatment of uterine leiomyoma.
Key Words
- Berberine;
- uterus;
- leiomyomas;
- fibroids;
- anti-tumorigenic;
- antineoplastic;
- treatment
Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/wuh-berberine-uterine-leiomyoma-cells/
Uterine leiomyomas are benign smooth muscle cell tumors of the myometrium and are the most common pelvic tumors in women 1 ; 2. Leiomyomas affect up to 50% of women ages 35–49 years (3).
Symptoms of uterine leiomyomas include acute and chronic pelvic pain,
excessive vaginal bleeding, dyspareunia, iron-deficiency anemia,
miscarriage, and infertility 4 ; 5.
The estimated economic burden for uterine leiomyomas in the United
States is large, with estimates ranging from $5.9 to 34.4 billion yearly
(6). Currently, there are no approved effective long-term medicinal treatments for these tumors.
Berberine
(BBR), a natural alkaloid isolated from a number of important medicinal
plant species such as Berberis aristata and Berberis aquifolium, is a
traditional Chinese herb with antibacterial (7), antihypertensive (8), anti-inflammatory (9), antidiabetic (10), and antihyperlipidemic (11)
effects. BBR has also been used for many years in North American folk
medicine to treat subacute and chronic inflammatory conditions including
gastric disorders, respiratory diseases, and cancer (12).
Recently, BBR has been shown to be effective in inhibiting the growth
of a variety of human cancers, including melanoma, lung cancer,
neuroblastoma, colonic carcinoma, breast cancer, and hepatocellular
carcinoma 13; 14; 15; 16; 17 ; 18.
The
antineoplastic effects of BBR are manifested both in vitro and in vivo,
as assessed by suppression of tumor cell proliferation, induction of
tumor cell apoptosis, and inhibition of both tumor invasion and
metastasis (19).
Molecular mechanisms for the antineoplastic properties of BBR involve
[1] p53 dependent cell-cycle arrests at G0/G1, G1, and/or G2/M and
suppressed expression of cyclins (e.g., cyclin B, D, E) and
cyclin-dependent kinases (e.g., CDK 2, 4, 6); [2] modulation of the
mitochondria/caspase-dependent and/or Fas/FasL signaling pathways,
resulting in alterations in the ratio of anti-apoptotic (Bcl-2 proper,
Bcl-XL) and proapoptotic (Bax, Bid) members of the Bcl-2 family
proteins; [3] changes in other cell signaling pathways including the
Ros, JNK, PKC, ERK, and ATF3 pathways; and [4] inducing apoptosis via
positive or negative regulation of various cytokines functioning in the
cellular network, including the up-regulation of GADD153, the inhibition
of cyclooxygenase-2 (COX-2) and Mcl-1, and the down-regulation of
nucleolar phosphoprotein nucleophosmin/B23 and telomerase (see review
12).
Collectively,
these mechanisms suggest that BBR may be a promising candidate for
clinical use in certain neoplastic growths. Consequently, we have
hypothesized that BBR will have a similar effect on normal and
leiomyomatous myometrial cells. To test this hypothesis, human uterine
leiomyoma (UtLM) and normal uterine smooth muscle cell (UtSMC) lines
were treated with BBR, and their proliferation, apoptosis, and
expression of related genes was determined.
Materials and methods
Cell Culture
Immortalized
UtLM and normal (UtSMC) human myometrial cell lines were provided by
Dr. Ayman Al-Hendy, from cells originally generated by Dr. Darlene Dixon
(20)
via transfection with human telomerase gene. Cells were maintained in
smooth muscle growth medium-2 (SmBM; catalog no. CC-3181, Lonza)
containing 5% fetal bovine serum (FBS) and supplemented with SmBM
singlequots (catalog no. CC-4149).This SmBM singlequot contains hEGF,
insulin, hFGF-B, and gentamicin/amphotericin-B. For the BBR stimulation
experiments, BBR was directly added to maintain medium.
For the 17β-estradiol (E2; Sigma, catalog no. E2758) stimulation experiments, cells were grown in serum-free SmBM for 24 hours and then treated with E2 and/or BBR in SmBM containing 1% FBS. For the progesterone (P4;
Sigma, catalog no. P8783) stimulation experiments, cells were grown in
serum-free Dulbecco's modified Eagle medium (DMEM) for 48 hours and then
treated with P4 and/or BBR in the same medium.
Cell Proliferation (MTS) Assay
Cell
proliferation was determined by using the CellTiter 96 Cell
Proliferation MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]
Assay kit (Promega). Experiments were conducted in 96-well plates with
5,000 cells/well initially. After treatment for 72 hours, cells were
washed twice using phosphate-buffered saline (PBS) and incubated in
100 μL per well of SmBM or DMEM (in P4 stimulation
experiment). Twenty microliters of CellTiter 96 solution was added to
each well. Absorbance was determined with a microplate reader at 490 nm.
Real-time Quantitative PCR (qPCR)
Total
RNA was extracted using the miRACLE Isolation Kit (Jinfiniti
Biosciences). First-strand cDNA of mRNA was synthesized using the High
Capacity cDNA Reverse transcription Kit (Applied Biosystems). Real-time
quantitative PCR was performed by using an iTag Universal SYBR Green
Supermix (Bio-Rad Laboratories, Inc.) on an Applied Biosystems 7300
real-time PCR system. Primers for two-cell proliferation markers (MKI67 [21] and PCNA [22]), six G2/M phase-related genes (cyclin A1, cyclin B1, P21, P53, cyclin-dependent kinase 1 [CDK1], and cyclin-dependent kinase 4 [CDK4]) 23; 24 ; 25, and three genes that are typically overexpressed and play important roles in the pathogenesis of uterine leiomyomas (pituitary tumor-transforming gene-1 [PTTG-1], E2F transcription factor 1 [E2F1], and cyclooxygenase-2 [COX-2]) 26; 27 ; 28 were purchased from www.realtimeprimers.com. β-Actin was used as an internal control. Relative fold change of targets genes expression was calculated by using the 2−ΔΔCt method.
Cell Cycle Analysis
UtLM
cells were plated in six-well plates with culture medium. Cells were
treated with various concentrations (0, 10, 20, and 50 μM) of BBR
(catalog no. B3251, Sigma-Aldrich) for 24 hours. The cells were then
collected and fixed in cold 70% ethanol at 4°C. After washing, the cells
were subsequently treated with 50 mg/mL propidium iodide (PI) and
100 mg/mL RNaseA for 30 minutes in the dark and subjected to
flow-cytometric analysis to determine the percentage of cells in
specific phases of the cell cycle (subG1, G0/G1, S, and G2/M).
Flow-cytometry was performed in the Georgia Regents University campus
flow-cytometry core facility by using FACSCalibur Analyzers (Becton
Dickinson) equipped with a 488-nm argon laser.
Annexin V Staining
The
ability of annexin V to specifically bind phosphatidylserine (PS) is
widely used in cellular biology as a method to detect apoptotic cells (29).
In normal viable cells, PS is located on the cytoplasmic surface of the
cell membrane. However, PS will translocate from the inner to the outer
leaflet of the membrane in the intermediate stages of apoptosis (30). This process exposes PS to the external cellular environment, where it can be detected.
To
detect apoptosis, UtLM cells were plated on eight-well chamber slides
incubated in culture medium overnight. Cells were then treated with
20 μM BBR for 24 hours. After incubation, cells were washed with cold
PBS and incubated in annexin V binding buffer (10 mM HEPES, 140 mM NaCl,
2.5 mM CaCl2) containing 5 μL of annexin V Alexa Fluor 488
conjugate (catalog no. A13201, Life Technologies) for 15 minutes at room
temperature. After incubation, cells were washed once with annexin V
binding buffer. The slide was mounted with Aqueous Mounting Medium with
anti-Fading agents (catalog no. M01, Biomeda Corporation) and examined
with fluorescent inverted microscopy (catalog no. CKX41, Olympus) at
488 nM.
Statistical Analysis
Comparisons
of multiple groups were carried out by analysis of variance (ANOVA)
followed by a post-test by using the Tukey (among groups) and Dunnett
(compared with control group) tests (XLSTAT Software). P<.05
was considered statistically significant. All experiments were repeated
3 times (n = 3). All values are presented as mean ± SEM. In Figure 1,
after ANOVA analysis, data were separated into three different groups
(a, b, and c). The differences among the three groups were significant (P<.01).
- Figure 1.BBR blocks the E2- and P4-induced proliferation of UtLM cells. UtLM cells were incubated with BBR and/or E2 (A) or P4 (B) at the indicated concentrations for 72 hours. Cell viability was determined by the MTS assay. The cell survival rate of untreated cells was considered as 100%. After ANOVA analysis, data were separated into three different groups (a, b, and c). The differences among the three groups were significant (P<.01). No significant differences were found between treatments inside one group. Data are shown as means ± SE; n = 3.
Results
BBR Inhibits the Proliferation and Induces Cell Cycle Arrest in UtLM Cells
BBR treatment significantly reduced UtLM cell viability in a dose-dependent manner (Fig. 2A
and 1B) and inhibited UtLM cell growth by approximately 34%, 66%, and
67% at concentrations of 10, 20, and 50 μM, respectively. BBR treatment
also significantly inhibited the expression of the cell proliferation
marker MKI67 ( Fig. 2C)
in a dose-dependent manner (by approximately 70%, 90%, and 99% at BBR
concentrations of 10, 20, and 50 μM, respectively). Alternatively, the
expression of PCNA decreased by approximately 40%, regardless of BBR dose.
- Figure 2.BBR inhibits cell proliferation and induces cell cycle arrest in UtLM and UtSMC cells. UtLM and UtSMC cells were incubated with BBR at the indicated concentrations for 72 and 24 hours for cell proliferation assay and cell cycle test. (A) Bright field pictures (×100) were taken after treatment. (B) Cell viability was determined by MTS assay; the cell survival rate of untreated cells was considered as 100%. (C) The gene expression of the proliferation markers, MKI67 and PCNA, after BBR treatment is seen. (D) Cells were fixed and stained with PI and analyzed by a flow-cytometer. Quantitation of the PI staining data is presented as the cell cycle distribution percentages. (E) Changes in the expression of cyclin A1 and cyclin B1 with BBR treatment. (F) Changes in the expression of p21 and p53 with BBR treatment. (G) Changes in the expression of CDK1 and CDK4 with BBR treatment. Data are shown as means ± SE; n = 3. ∗P<.05 and ∗∗P<.01 vs. 0 μM of BBR.
In UtSMC cells, BBR (50 μM) did not have a significant effect on cell proliferation (Fig. 2A and 2B). BBR in UtSMC cells inhibited MKI67 and PCNA expression by 40% and 20%, respectively ( Fig. 2C).
BBR treatment induced the accumulation of UtLM cells in the G2/M phase (50% at 50 μM BBR; Fig. 2D),
accompanied by a decrease in the number of cells in G0/G1. In addition,
BBR inhibited the expression of the G2/M phase-related genes cyclin A1 (by approximately 20%, 40%, and 55%) and cyclin B1 (by approximately 40%, 80%, and 95%), while increasing the expression of P21 (by approximately 4-, 5-, and 9-fold), at concentrations of 10, 20, and 50 μM, respectively ( Fig. 2E and 1F). In UtLM cells, BBR also increased the expression of the G2/M phase-related gene P53 by about 50%, although not in a dose-dependent manner ( Fig. 2F). As expected, BBR inhibited the expression of CDK1 (by approximately 40%, 80%, and 95%) in a dose-dependent fashion ( Fig. 2G); alternatively, it increased CDK4 expression by approximately 30%, regardless of BBR dose ( Fig. 2G).
In UtSMC cells, BBR (50 μM) led to only 15% of cells being arrested in the G2/M phase (Fig. 2D) and had no effect on cyclin A1 and cyclin B1 expression ( Fig. 2E). BBR treatment (50 μM) increased expression of P21 (3-fold) in UtSMC cells but had no effect on P53 expression ( Fig. 2F) or CDK1 and CDK4 expression ( Fig. 2G).
BBR Inhibits the Expression of Genes That are Uniquely Overexpressed in UtLM Cells
We examined the expression of three genes, PTTG-1, E2F1, and COX-2,
which are typically overexpressed and play important roles in the
pathogenesis of uterine leiomyomas. Our results indicate that BBR, in a
dose-dependent fashion, reduces the expression of each of these genes in
UtLM cells ( Fig. 3). Alternatively, in UtSMC cells, BBR did not seem to have an effect on PTTG1 expression but did decrease E2F1 and COX-2 expression ( Fig. 3).
- Figure 3.BBR down-regulates PTTG-1, E2F1, and COX-2 expression in UtLM and UtSMC cells. UtLM and UtSMC cells were incubated with BBR at the indicated concentrations for 24 hours. Gene expression was determined by real-time RT-PCR. Data are shown as means ± SE; n = 3. ∗P<.05 and ∗∗P<.01 vs. 0 μM of BBR.
BBR Induces UtLM Cell Apoptosis
Induction of apoptosis by BBR was evaluated by annexin V staining and BAX
expression. BBR (20 μM) clearly induced apoptosis in UtLM cells with
positive annexin V staining after 24 hours; no such effect was observed
in vehicle-treated cells ( Fig. 4A). Furthermore, BAX expression in UtLM cells was induced by BBR in a dose-dependent manner, peaking at 20 μM (approximately 2.5 fold; Fig. 4B). Alternatively, in UtSMC cells, BBR (20 μM) did not induce apoptosis, with negative annexin V staining after 24 hours (Fig. 4A), and induced the expression of BAX in these cells by just 50% ( Fig. 4B).
- Figure 4.BBR induces apoptosis in UtLM and UtSMC cells. UtLM and UtSMC cells were incubated with BBR at the indicated concentrations for 24 hours. (A) Annexin V staining of UtLM cells (×100). (B) Changes in BAX gene expression with BBR treatment. Data are shown as means ± SE; n = 3. ∗∗P<.01 vs. 0 μM of BBR.
BBR Blocks E2- and P4-stimulated Cell Proliferation in UtLM Cells
To determine whether BBR inhibits the proliferative effects of E2, UtLM cells were incubated with BBR and/or E2 for 72 hours (Fig. 1A). As expected, E2
significantly stimulated the growth of UtLM cells by approximately 40%
and 60%, at concentrations of 10 and 100 nM in SmBM containing 1% FBS,
respectively. In turn, BBR (20 and 50 μM) similarly inhibited UtLM cell
proliferation and totally blocked the E2-induced proliferation of these cells.
P4 significantly stimulated the growth of UtLM cells by approximately 30% at concentrations of 100 nM in DMEM (Fig. 1B). Again, to determine whether BBR inhibits the proliferative effects of P4, UtLM cells were incubated with BBR and/or P4 for 72 hours. Our results indicate that 50 μM of BBR totally blocked the P4 (100 nM)-induced proliferation of these cells (Fig. 1B).
Discussion
BBR
has antineoplastic activities in a variety of human cancers. However,
the effects of BBR on UtLM cells have not been previously investigated.
We now present evidence that BBR inhibits human leiomyoma cell
proliferation. Similar to other human cancers, BBR inhibition of human
leiomyoma cells was generally dose dependent and was mediated through
the inhibition of cellular proliferation and apoptosis. Treatment of
leiomyoma cells with BBR inhibited cell proliferation by approximately
60%. BBR also significantly inhibited the expression of the cell
proliferation markers MKI67 and PCNA.
In
addition, BBR induced cell cycle arrest in the G2/M phase in leiomyoma
cells. We found that the expression of cell cycle G2/M phase-related
genes, including cyclin A1, cyclin B1, p21, p53, CDK1, and CDK4, was altered by BBR treatment. The cyclin B1-CDK1 complex is recognized as an M phase-promoting factor (MPF) (23), and disruption of cyclin B1 prevents mitotic entry (24). Cyclin A1 is a trigger for MPF activation, and knockdown of cyclin A1 induces cell cycle arrest in the G2 phase (24). Our results indicated that BBR significantly reduces cyclin A1, cyclin B1, and CDk1 gene expression in leiomyoma cells. Treatment with BBR also increased the expression of p53 and p21; and overexpression of p53 induces cell cycle arrest at G2/M phase and is associated with high expression of p21 (25).
Alternatively, in normal UtSMCs, BBR did not have a significant effect
on cell proliferation, with modest to no effects on the expression of
related genes. Together these data suggest that the effect of BBR on
cell cycle arrest in the G2/M phase in leiomyoma cells is mediated by
regulating the expression of cyclin A1, cyclin B1, CDK1, p21, and p53.
To
determine whether BBR treatment induces apoptosis in leiomyoma cells,
we assessed the presence of annexin V by histochemistry and the
expression of BAX. Leiomyoma cells exhibited increased annexin V staining after treatment with 20 μM BBR for 48 hours compared with control. Bax is a proapoptotic member of the Bcl-2 family that induces apoptosis in cancer cells (31), and BBR was able to induce the expression of BAX in leiomyoma cells. In addition, BBR treatment also stimulated p53 expression. Overexpression of p53 causes apoptosis in cancer cells (32) and is BAX dependent (33).
Alternatively, in normal UtSMCs BBR did not have a significant effect
on cell apoptosis, with limited effects on the expression of related
genes. These data suggest that BBR-induced apoptosis of leiomyoma cells
is, at least in part, mediated through the p53-dependent cell death pathway.
Estrogen and P are important uterine growth factors that induce human leiomyoma (and normal myometrial) cell proliferation 34 ; 35, a fact confirmed by our studies. However, BBR treatment was able to block E2- and P4-induced
cell proliferation in leiomyoma cells, which suggests that BBR may have
antiestrogenic or antiprogestin effects in these cells.
PTTG-1, or securin, is a novel proto-oncogene first discovered in the rat pituitary tumor cell line GH4 (36). Tsai et al. (26) reported a positive correlation between PTTG-1, bFGF, and PCNA in uterine leiomyomas and a positive feedback loop between PTTG-1
and bFGF in cultured leiomyoma cells. These investigators suggested
that related autocrine/paracrine cross talk may explain, to some extent,
why antiestrogen or antiprogesterone treatments fail to cause complete
regression of leiomyomas. Our data demonstrate that BBR reduced the
expression of PTTG-1 in culture medium containing bFGF, which
suggests that BBR may abrogate this positive feedback loop between
PTTG-1 and bFGF and thus cause regression of leiomyoma.
COX-2 is a critical enzyme that converts arachidonic acid into prostaglandin E2 (PGE2) and is commonly overexpressed in many solid tumors, including colorectal, breast, prostate, and ovarian neoplasms (37). Increased expression of COX-2 and the associated PGE2 production have been demonstrated to significantly enhance carcinogenesis (38). Ke et al. (28) reported that COX-2 expression was significantly up-regulated in uterine leiomyomas and that the inhibition of COX-2
activity significantly reduced the proliferation of the uterine
fibroids smooth muscle cells, which suggests that COX-2 is involved in
the pathogenesis of uterine leiomyomas. In turn, BBR has been reported
to induce cancer cell apoptosis and suppress cancer cell migration in
many neoplastic cell lines, including melanoma (39), non–small cell lung cancer (40), and oral cancer (41),
an effect mediated through the reduced expression of COX-2. Consistent
with these observations, our data indicate that BBR significantly
reduced COX-2 expression in leiomyoma cells, which suggests
that COX-2 may also play a role in mediating BBR-induced apoptosis in
human leiomyoma cells.
To
determine whether BBR was cytotoxic in this model, we treated normal
UtSMCs with different doses of BBR. We found that the highest dose of
BBR used (50 μM), which had caused a 67% inhibition in the proliferation
of leiomyoma cells, did not significantly inhibit the proliferation of
normal myometrial cells. BBR treatment also had no or very little effect
on cycle, proliferation, or apoptosis-related genes expression. These
results suggest that BBR appears to have little or no cytotoxicity on
UtSMCs. The cytotoxic effects of BBR specific in cancer cell lines but
not in normal cells have also been reported in hepatoma (42), prostate cancer (43), colon cancer 44 ; 45, and breast cancer (15). In addition, BBR reduced the expression of E2F1 and COX-2 in normal myometrial cells, which suggests that BBR could prevent the transformation of normal myometrial cells.
We
should recognize the limitations of this study. First, these
experiments used cell lines and ex vivo tissues, which may not reflect
the clinical condition. Although the two transformed cell lines used,
UtLM and UtSMC, come from humans originally and demonstrate no
phenotypic alteration from the parental cell types (20),
in vitro study with these cell lines may not reveal all activities of
BBR in vivo. Therefore, studies in vivo will be necessary to confirm our
findings. Second, the BBR concentrations in cell culture may not
reflect the actual concentration of BBR experienced by tissues in vivo
with therapeutic administration, and again in vivo dose response studies
are needed.
In summary, our data demonstrate that BBR inhibits spontaneous and E2- or P4-induced
cell proliferation and induces apoptosis in UtLM cells but does not
demonstrate a significant cytotoxic effect in normal human UtSMCs. Taken
together, our results suggest that BBR could be a potential therapeutic
agent for the medical treatment of uterine leiomyomas.
References
- 1
- Uterine fibroids
- Lancet, 357 (2001), pp. 293–298
- | | |
- 2
- Medical management of fibroids
- Best Pract Res Clin Obstet Gynaecol, 22 (2008), pp. 655–676
- | | |
- 3
- High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence
- Am J Obstet Gynecol, 188 (2003), pp. 100–107
- |
- 4
- The effect of intramural fibroids without uterine cavity involvement on the outcome of IVF treatment: a systematic review and meta-analysis
- Hum Reprod, 25 (2010), pp. 418–429
- | |
- 5
- Image-guided thermal therapy of uterine fibroids
- Semin Ultrasound CT MR, 30 (2009), pp. 91–104
- | | |
- 6
- The estimated annual cost of uterine leiomyomata in the United States
- Am J Obstet Gynecol, 206 (2012), pp. 211.e1–211.e9
- |
- 7
- Evaluation of the antimicrobial mode of berberine by LC/ESI-MS combined with principal component analysis
- J Pharm Biomed Anal, 44 (2007), pp. 301–304
- | | |
- 8
- The antihypertensive effect of the berberine derivative 6-protoberberine in spontaneously hypertensive rats
- Pharmacology, 59 (1999), pp. 283–289
- | |
- 9
- The anti-inflammatory potential of berberine in vitro and in vivo
- Cancer Lett, 203 (2004), pp. 127–137
- | | |
- 10
- Anti-diabetic effects of cinnamaldehyde and berberine and their impacts on retinol-binding protein 4 expression in rats with type 2 diabetes mellitus
- Chin Med J (Engl), 121 (2008), pp. 2124–2128
- |
- 11
- Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins
- Nat Med, 10 (2004), pp. 1344–1351
- |
- 12
- Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations
- J Ethnopharmacol, 126 (2009), pp. 5–17
- | | |
- 13
- Berberine inhibits human neuroblastoma cell growth through induction of p53-dependent apoptosis
- Anticancer Res, 28 (2008), pp. 3777–3784
- 14
- Synthesis and structure-activity relationship of berberine analogues in LDLR up-regulation and AMPK activation
- Bioorg Med Chem, 20 (2012), pp. 6552–6558
- | | |
- 15
- Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway
- Eur J Pharmacol, 645 (2010), pp. 70–78
- | | |
- 16
- Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/beta-catenin signaling
- Int J Oncol, 41 (2012), pp. 292–298
- |
- 17
- Epithelial-to-mesenchymal transition markers to predict response of Berberine in suppressing lung cancer invasion and metastasis
- J Transl Med, 12 (2014), p. 22
- |
- 18
- Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft
- Phytomedicine, 21 (2014), pp. 340–347
- | | |
- 19
- A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs
- Anticancer Drugs, 20 (2009), pp. 757–769
- |
- 20
- Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics
- Lab Invest, 82 (2002), pp. 719–728
- | |
- 21
- MicroRNA-519d targets MKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703
- Cancer Lett, 307 (2011), pp. 182–190
- | | |
- 22
- Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation
- Ann Bot, 107 (2011), pp. 1127–1140
- | |
- 23
- From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner?
- J Cell Sci, 115 (2002), pp. 2461–2464
- |
- 24
- Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor
- Mol Biol Cell, 18 (2007), pp. 1861–1873
- 25
- p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts
- Proc Natl Acad Sci U S A, 92 (1995), pp. 8493–8497
- | |
- 26
- Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas
- J Clin Endocrinol Metab, 90 (2005), pp. 3715–3723
- | |
- 27
- An E2F1-dependent gene expression program that determines the balance between proliferation and cell death
- Cancer Cell, 13 (2008), pp. 11–22
- | | |
- 28
- High expression of cyclooxygenase-2 in uterine fibroids and its correlation with cell proliferation
- Eur J Obstet Gynecol Reprod Biol, 168 (2013), pp. 199–203
- | | |
- 29
- Colourful death: six-parameter classification of cell death by flow cytometry—dead cells tell tales
- Autoimmunity, 46 (2013), pp. 336–341
- | |
- 30
- Surface code—biophysical signals for apoptotic cell clearance
- Phys Biol, 10 (2013), p. 065007
- 31
- Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer
- Cancer Res, 61 (2001), pp. 186–191
- |
- 32
- Control of apoptosis by p53
- Oncogene, 22 (2003), pp. 9030–9040
- | |
- 33
- Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis
- Science, 303 (2004), pp. 1010–1014
- | |
- 34
- Human uterine smooth muscle and leiomyoma cells differ in their rapid 17beta-estradiol signaling: implications for proliferation
- Endocrinology, 150 (2009), pp. 2436–2445
- | |
- 35
- The role of progesterone signaling in the pathogenesis of uterine leiomyoma
- Mol Cell Endocrinol, 358 (2012), pp. 223–231
- | | | |
- 36
- Isolation and characterization of a pituitary tumor-transforming gene (PTTG)
- Mol Endocrinol, 11 (1997), pp. 433–441
- | |
- 37
- HER2 and COX2 expression in human prostate cancer
- Eur J Cancer, 40 (2004), pp. 50–55
- | | |
- 38
- The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment
- Carcinogenesis, 30 (2009), pp. 377–386
- | |
- 39
- Berberine, an isoquinoline alkaloid, inhibits melanoma cancer cell migration by reducing the expressions of cyclooxygenase-2, prostaglandin E(2) and prostaglandin E(2) receptors
- Carcinogenesis, 32 (2011), pp. 86–92
- | |
- 40
- Berberine targets AP-2/hTERT, NF-kappaB/COX-2, HIF-1alpha/VEGF and cytochrome-c/caspase signaling to suppress human cancer cell growth
- PLoS One, 8 (2013), p. e69240
- 41
- Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells
- In Vivo, 19 (2005), pp. 247–252
- |
- 42
- Berberine inhibits human hepatoma cell invasion without cytotoxicity in healthy hepatocytes
- PLoS One, 6 (2011), p. e21416
- 43
- Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells
- Mol Cancer Ther, 5 (2006), pp. 296–308
- | |
- 44
- Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor
- PLoS One, 7 (2012), p. e36418
- 45
- The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells
- Eur J Pharmacol, 688 (2012), pp. 14–21
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