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Berberine inhibits the proliferation of human uterine leiomyoma cells
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
Berberine inhibits the proliferation of human uterine leiomyoma cells Hsiao-Li Wu, M.S., M.B.A.,a Tung-Yueh Chuang, Ph.D.,a Ayman Al-Hendy, M.D., Ph.D.,a Michael P. Diamond, M.D.,a Ricardo Azziz, M.D., M.B.A., M.P.H.,a,b and Yen-Hao Chen, Ph.D.a a
Department of Obstetrics/Gynecology and b Department of Medicine, Georgia Regents University, Augusta, Georgia
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 mM), [2] BBR (20 and 50 mM) and/or 17b-estradiol (E2; 10 and 100 nM), and [3] BBR (20 and 50 mM) 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 mM) 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. (Fertil SterilÒ 2015;-: Use your smartphone -–-. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Berberine, uterus, leiomyomas, fibroids, anti-tumorigenic, antineoplastic, and connect to the treatment Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/wuh-berberine-uterine-leiomyoma-cells/
U
terine 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), antiinflammatory (9), antidiabetic (10), and antihyperlipidemic (11) effects.
Received October 3, 2014; revised and accepted January 7, 2015. H.-L.W. has nothing to disclose. T.-Y.C. has nothing to disclose. A.A.-H. has nothing to disclose. M.P.D. reports grants from Abbvie and Bayer unrelated to the submitted work. R.A. has nothing to disclose. Y.-H.C. has nothing to disclose. This work was supported by Georgia Regents University research funds (to Y.-H.C). Reprint requests: Yen-Hao Chen, Ph.D., Georgia Regents University, 1120 15th Street, CA-2020, Augusta, Georgia 30912 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.01.010 VOL. - NO. - / - 2015
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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 (13–18). 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 1
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 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 17b-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 serumfree Dulbecco's modified Eagle medium (DMEM) for 48 hours and then treated with P4 and/or BBR in the same medium.
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]) (23–25), 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]) (26–28) were purchased from www.realtimeprimers.com. b-Actin was used as an internal control. Relative fold change of targets genes expression was calculated by using the 2DDCt 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 mM) 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
Cell proliferation was determined by using the CellTiter 96 Cell Proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-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 mL 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.
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 mM 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 mL 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 antiFading agents (catalog no. M01, Biomeda Corporation) and examined with fluorescent inverted microscopy (catalog no. CKX41, Olympus) at 488 nM.
Real-time Quantitative PCR (qPCR)
Statistical Analysis
Total RNA was extracted using the miRACLE Isolation Kit (Jinfiniti Biosciences). First-strand cDNA of mRNA was synthesized
Comparisons of multiple groups were carried out by analysis of variance (ANOVA) followed by a post-test by using the
Cell Proliferation (MTS) Assay
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Fertility and Sterility® 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 ¼ 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).
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 mM, 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 mM, respectively). Alternatively, the expression of PCNA decreased by approximately 40%, regardless of BBR dose. In UtSMC cells, BBR (50 mM) 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 mM 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 mM, 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 mM) 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 mM) 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).
BBR Induces UtLM Cell Apoptosis Induction of apoptosis by BBR was evaluated by annexin V staining and BAX expression. BBR (20 mM) 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 mM (approximately 2.5 fold; Fig. 4B). Alternatively, in UtSMC cells, BBR (20 mM) did not induce apoptosis, with
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 ¼ 3. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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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 ¼ 3. *P<.05 and **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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FIGURE 2 Continued
Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
negative annexin V staining after 24 hours (Fig. 4A), and induced the expression of BAX in these cells by just 50% (Fig. 4B).
erative effects of P4, UtLM cells were incubated with BBR and/ or P4 for 72 hours. Our results indicate that 50 mM of BBR totally blocked the P4 (100 nM)-induced proliferation of these cells (Fig. 1B).
BBR Blocks E2- and P4-stimulated Cell Proliferation in UtLM Cells
DISCUSSION
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 mM) 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 prolif-
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.
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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 ¼ 3. *P<.05 and **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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 mM 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 BBRinduced 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 6
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 mM), which had caused a 67% inhibition in the proliferation of leiomyoma VOL. - NO. - / - 2015
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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 ¼ 3. **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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 VOL. - NO. - / - 2015
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 7
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 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. 2. 3.
4.
5. 6.
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
8
Stewart EA. Uterine fibroids. Lancet 2001;357:293–8. Sankaran S, Manyonda IT. Medical management of fibroids. Best Pract Res Clin Obstet Gynaecol 2008;22:655–76. Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence. Am J Obstet Gynecol 2003;188:100–7. Sunkara SK, Khairy M, El-Toukhy T, Khalaf Y, Coomarasamy A. The effect of intramural fibroids without uterine cavity involvement on the outcome of IVF treatment: a systematic review and meta-analysis. Hum Reprod 2010;25: 418–29. Shen SH, Fennessy F, McDannold N, Jolesz F, Tempany C. Image-guided thermal therapy of uterine fibroids. Semin Ultrasound CT MR 2009;30:91–104. Cardozo ER, Clark AD, Banks NK, Henne MB, Stegmann BJ, Segars JH. The estimated annual cost of uterine leiomyomata in the United States. Am J Obstet Gynecol 2012;206:211.e1–9. Yi ZB, Yan Y, Liang YZ, Bao Z. Evaluation of the antimicrobial mode of berberine by LC/ESI-MS combined with principal component analysis. J Pharm Biomed Anal 2007;44:301–4. Liu JC, Chan P, Chen YJ, Tomlinson B, Hong SH, Cheng JT. The antihypertensive effect of the berberine derivative 6-protoberberine in spontaneously hypertensive rats. Pharmacology 1999;59:283–9. Kuo CL, Chi CW, Liu TY. The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett 2004;203:127–37. Zhang W, Xu YC, Guo FJ, Meng Y, Li ML. 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) 2008; 121:2124–8. Kong W, Wei J, Abidi P, Lin M, Inaba S, Li C, et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nat Med 2004;10:1344–51. Tang J, Feng Y, Tsao S, Wang N, Curtain R, Wang Y. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J Ethnopharmacol 2009;126:5–17. Choi MS, Yuk DY, Oh JH, Jung HY, Han SB, Moon DC, et al. Berberine inhibits human neuroblastoma cell growth through induction of p53dependent apoptosis. Anticancer Res 2008;28:3777–84. Wang YX, Kong WJ, Li YH, Tang S, Li Z, Li YB, et al. Synthesis and structureactivity relationship of berberine analogues in LDLR up-regulation and AMPK activation. Bioorg Med Chem 2012;20:6552–8. Patil JB, Kim J, Jayaprakasha GK. Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway. Eur J Pharmacol 2010;645:70–8. Wu K, Yang Q, Mu Y, Zhou L, Liu Y, Zhou Q, et al. Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/beta-catenin signaling. Int J Oncol 2012;41:292–8.
17.
18.
19.
20.
21.
22. 23. 24.
25.
26.
27.
28.
29.
30.
31.
32. 33.
34.
35. 36. 37. 38.
39.
Qi HW, Xin LY, Xu X, Ji XX, Fan LH. Epithelial-to-mesenchymal transition markers to predict response of Berberine in suppressing lung cancer invasion and metastasis. J Transl Med 2014;12:22. Mittal A, Tabasum S, Singh RP. Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft. Phytomedicine 2014;21:340–7. Sun Y, Xun K, Wang Y, Chen X. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs. Anticancer Drugs 2009;20:757–69. Carney SA, Tahara H, Swartz CD, Risinger JI, He H, Moore AB, et al. Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics. Lab Invest 2002;82:719–28. Hou YY, Cao WW, Li L, Li SP, Liu T, Wan HY, et al. MicroRNA-519d targets MKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703. Cancer Lett 2011;307:182–90. Strzalka W, Ziemienowicz A. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot 2011;107:1127–40. Doree M, Hunt T. From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J Cell Sci 2002;115:2461–4. Fung TK, Ma HT, Poon RY. Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor. Mol Biol Cell 2007;18:1861–73. Agarwal ML, Agarwal A, Taylor WR, Stark GR. 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 1995;92:8493–7. Tsai SJ, Lin SJ, Cheng YM, Chen HM, Wing LY. Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas. J Clin Endocrinol Metab 2005;90:3715–23. Hallstrom TC, Mori S, Nevins JR. An E2F1-dependent gene expression program that determines the balance between proliferation and cell death. Cancer Cell 2008;13:11–22. Ke X, Dou F, Cheng Z, Dai H, Zhang W, Qu X, et al. High expression of cyclooxygenase-2 in uterine fibroids and its correlation with cell proliferation. Eur J Obstet Gynecol Reprod Biol 2013;168:199–203. Munoz LE, Maueroder C, Chaurio R, Berens C, Herrmann M, Janko C. Colourful death: six-parameter classification of cell death by flow cytometry— dead cells tell tales. Autoimmunity 2013;46:336–41. Biermann M, Maueroder C, Brauner JM, Chaurio R, Janko C, Herrmann M, et al. Surface code—biophysical signals for apoptotic cell clearance. Phys Biol 2013;10:065007. Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, et al. Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 2001;61:186–91. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 2003;22: 9030–40. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 2004;303:1010–4. Nierth-Simpson EN, Martin MM, Chiang TC, Melnik LI, Rhodes LV, Muir SE, et al. Human uterine smooth muscle and leiomyoma cells differ in their rapid 17beta-estradiol signaling: implications for proliferation. Endocrinology 2009;150:2436–45. Kim JJ, Sefton EC. The role of progesterone signaling in the pathogenesis of uterine leiomyoma. Mol Cell Endocrinol 2012;358:223–31. Pei L, Melmed S. Isolation and characterization of a pituitary tumortransforming gene (PTTG). Mol Endocrinol 1997;11:433–41. Edwards J, Mukherjee R, Munro AF, Wells AC, Almushatat A, Bartlett JM. HER2 and COX2 expression in human prostate cancer. Eur J Cancer 2004;40:50–5. Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C, et al. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377–86. Singh T, Vaid M, Katiyar N, Sharma S, Katiyar SK. 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 2011;32:86–92.
VOL. - NO. - / - 2015
Fertility and Sterility® 40.
41.
42.
Fu L, Chen W, Guo W, Wang J, Tian Y, Shi D, et al. 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 2013;8:e69240. Kuo CL, Chi CW, Liu TY. Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells. In Vivo 2005;19:247–52. Liu B, Wang G, Yang J, Pan X, Yang Z, Zang L. Berberine inhibits human hepatoma cell invasion without cytotoxicity in healthy hepatocytes. PLoS One 2011;6:e21416.
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43.
44.
45.
Mantena SK, Sharma SD, Katiyar SK. Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol Cancer Ther 2006;5:296–308. Wang L, Liu L, Shi Y, Cao H, Chaturvedi R, Calcutt MW, et al. Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor. PLoS One 2012;7:e36418. Chidambara Murthy KN, Jayaprakasha GK, Patil BS. The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells. Eur J Pharmacol 2012;688:14–21.
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Berberine inhibits the proliferation of human uterine leiomyoma cells Hsiao-Li Wu, M.S., M.B.A.,a Tung-Yueh Chuang, Ph.D.,a Ayman Al-Hendy, M.D., Ph.D.,a Michael P. Diamond, M.D.,a Ricardo Azziz, M.D., M.B.A., M.P.H.,a,b and Yen-Hao Chen, Ph.D.a a
Department of Obstetrics/Gynecology and b Department of Medicine, Georgia Regents University, Augusta, Georgia
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 mM), [2] BBR (20 and 50 mM) and/or 17b-estradiol (E2; 10 and 100 nM), and [3] BBR (20 and 50 mM) 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 mM) 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. (Fertil SterilÒ 2015;-: Use your smartphone -–-. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Berberine, uterus, leiomyomas, fibroids, anti-tumorigenic, antineoplastic, and connect to the treatment Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/wuh-berberine-uterine-leiomyoma-cells/
U
terine 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), antiinflammatory (9), antidiabetic (10), and antihyperlipidemic (11) effects.
Received October 3, 2014; revised and accepted January 7, 2015. H.-L.W. has nothing to disclose. T.-Y.C. has nothing to disclose. A.A.-H. has nothing to disclose. M.P.D. reports grants from Abbvie and Bayer unrelated to the submitted work. R.A. has nothing to disclose. Y.-H.C. has nothing to disclose. This work was supported by Georgia Regents University research funds (to Y.-H.C). Reprint requests: Yen-Hao Chen, Ph.D., Georgia Regents University, 1120 15th Street, CA-2020, Augusta, Georgia 30912 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.01.010 VOL. - NO. - / - 2015
discussion forum for this article now.*
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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 (13–18). 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 1
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 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 17b-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 serumfree Dulbecco's modified Eagle medium (DMEM) for 48 hours and then treated with P4 and/or BBR in the same medium.
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]) (23–25), 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]) (26–28) were purchased from www.realtimeprimers.com. b-Actin was used as an internal control. Relative fold change of targets genes expression was calculated by using the 2DDCt 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 mM) 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
Cell proliferation was determined by using the CellTiter 96 Cell Proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-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 mL 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.
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 mM 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 mL 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 antiFading agents (catalog no. M01, Biomeda Corporation) and examined with fluorescent inverted microscopy (catalog no. CKX41, Olympus) at 488 nM.
Real-time Quantitative PCR (qPCR)
Statistical Analysis
Total RNA was extracted using the miRACLE Isolation Kit (Jinfiniti Biosciences). First-strand cDNA of mRNA was synthesized
Comparisons of multiple groups were carried out by analysis of variance (ANOVA) followed by a post-test by using the
Cell Proliferation (MTS) Assay
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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 mM, 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 mM, respectively). Alternatively, the expression of PCNA decreased by approximately 40%, regardless of BBR dose. In UtSMC cells, BBR (50 mM) 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 mM 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 mM, 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 mM) 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 mM) 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).
BBR Induces UtLM Cell Apoptosis Induction of apoptosis by BBR was evaluated by annexin V staining and BAX expression. BBR (20 mM) 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 mM (approximately 2.5 fold; Fig. 4B). Alternatively, in UtSMC cells, BBR (20 mM) did not induce apoptosis, with
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 ¼ 3. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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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 ¼ 3. *P<.05 and **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
negative annexin V staining after 24 hours (Fig. 4A), and induced the expression of BAX in these cells by just 50% (Fig. 4B).
erative effects of P4, UtLM cells were incubated with BBR and/ or P4 for 72 hours. Our results indicate that 50 mM of BBR totally blocked the P4 (100 nM)-induced proliferation of these cells (Fig. 1B).
BBR Blocks E2- and P4-stimulated Cell Proliferation in UtLM Cells
DISCUSSION
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 mM) 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 prolif-
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.
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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 ¼ 3. *P<.05 and **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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 mM 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 BBRinduced 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 6
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 mM), which had caused a 67% inhibition in the proliferation of leiomyoma VOL. - NO. - / - 2015
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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 ¼ 3. **P<.01 vs. 0 mM of BBR. Wu. Berberine inhibits human leiomyoma. Fertil Steril 2015.
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 VOL. - NO. - / - 2015
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 7
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 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. 2. 3.
4.
5. 6.
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
8
Stewart EA. Uterine fibroids. Lancet 2001;357:293–8. Sankaran S, Manyonda IT. Medical management of fibroids. Best Pract Res Clin Obstet Gynaecol 2008;22:655–76. Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence. Am J Obstet Gynecol 2003;188:100–7. Sunkara SK, Khairy M, El-Toukhy T, Khalaf Y, Coomarasamy A. The effect of intramural fibroids without uterine cavity involvement on the outcome of IVF treatment: a systematic review and meta-analysis. Hum Reprod 2010;25: 418–29. Shen SH, Fennessy F, McDannold N, Jolesz F, Tempany C. Image-guided thermal therapy of uterine fibroids. Semin Ultrasound CT MR 2009;30:91–104. Cardozo ER, Clark AD, Banks NK, Henne MB, Stegmann BJ, Segars JH. The estimated annual cost of uterine leiomyomata in the United States. Am J Obstet Gynecol 2012;206:211.e1–9. Yi ZB, Yan Y, Liang YZ, Bao Z. Evaluation of the antimicrobial mode of berberine by LC/ESI-MS combined with principal component analysis. J Pharm Biomed Anal 2007;44:301–4. Liu JC, Chan P, Chen YJ, Tomlinson B, Hong SH, Cheng JT. The antihypertensive effect of the berberine derivative 6-protoberberine in spontaneously hypertensive rats. Pharmacology 1999;59:283–9. Kuo CL, Chi CW, Liu TY. The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett 2004;203:127–37. Zhang W, Xu YC, Guo FJ, Meng Y, Li ML. 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) 2008; 121:2124–8. Kong W, Wei J, Abidi P, Lin M, Inaba S, Li C, et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nat Med 2004;10:1344–51. Tang J, Feng Y, Tsao S, Wang N, Curtain R, Wang Y. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J Ethnopharmacol 2009;126:5–17. Choi MS, Yuk DY, Oh JH, Jung HY, Han SB, Moon DC, et al. Berberine inhibits human neuroblastoma cell growth through induction of p53dependent apoptosis. Anticancer Res 2008;28:3777–84. Wang YX, Kong WJ, Li YH, Tang S, Li Z, Li YB, et al. Synthesis and structureactivity relationship of berberine analogues in LDLR up-regulation and AMPK activation. Bioorg Med Chem 2012;20:6552–8. Patil JB, Kim J, Jayaprakasha GK. Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway. Eur J Pharmacol 2010;645:70–8. Wu K, Yang Q, Mu Y, Zhou L, Liu Y, Zhou Q, et al. Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/beta-catenin signaling. Int J Oncol 2012;41:292–8.
17.
18.
19.
20.
21.
22. 23. 24.
25.
26.
27.
28.
29.
30.
31.
32. 33.
34.
35. 36. 37. 38.
39.
Qi HW, Xin LY, Xu X, Ji XX, Fan LH. Epithelial-to-mesenchymal transition markers to predict response of Berberine in suppressing lung cancer invasion and metastasis. J Transl Med 2014;12:22. Mittal A, Tabasum S, Singh RP. Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft. Phytomedicine 2014;21:340–7. Sun Y, Xun K, Wang Y, Chen X. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs. Anticancer Drugs 2009;20:757–69. Carney SA, Tahara H, Swartz CD, Risinger JI, He H, Moore AB, et al. Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics. Lab Invest 2002;82:719–28. Hou YY, Cao WW, Li L, Li SP, Liu T, Wan HY, et al. MicroRNA-519d targets MKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703. Cancer Lett 2011;307:182–90. Strzalka W, Ziemienowicz A. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot 2011;107:1127–40. Doree M, Hunt T. From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J Cell Sci 2002;115:2461–4. Fung TK, Ma HT, Poon RY. Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor. Mol Biol Cell 2007;18:1861–73. Agarwal ML, Agarwal A, Taylor WR, Stark GR. 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 1995;92:8493–7. Tsai SJ, Lin SJ, Cheng YM, Chen HM, Wing LY. Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas. J Clin Endocrinol Metab 2005;90:3715–23. Hallstrom TC, Mori S, Nevins JR. An E2F1-dependent gene expression program that determines the balance between proliferation and cell death. Cancer Cell 2008;13:11–22. Ke X, Dou F, Cheng Z, Dai H, Zhang W, Qu X, et al. High expression of cyclooxygenase-2 in uterine fibroids and its correlation with cell proliferation. Eur J Obstet Gynecol Reprod Biol 2013;168:199–203. Munoz LE, Maueroder C, Chaurio R, Berens C, Herrmann M, Janko C. Colourful death: six-parameter classification of cell death by flow cytometry— dead cells tell tales. Autoimmunity 2013;46:336–41. Biermann M, Maueroder C, Brauner JM, Chaurio R, Janko C, Herrmann M, et al. Surface code—biophysical signals for apoptotic cell clearance. Phys Biol 2013;10:065007. Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, et al. Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 2001;61:186–91. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 2003;22: 9030–40. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 2004;303:1010–4. Nierth-Simpson EN, Martin MM, Chiang TC, Melnik LI, Rhodes LV, Muir SE, et al. Human uterine smooth muscle and leiomyoma cells differ in their rapid 17beta-estradiol signaling: implications for proliferation. Endocrinology 2009;150:2436–45. Kim JJ, Sefton EC. The role of progesterone signaling in the pathogenesis of uterine leiomyoma. Mol Cell Endocrinol 2012;358:223–31. Pei L, Melmed S. Isolation and characterization of a pituitary tumortransforming gene (PTTG). Mol Endocrinol 1997;11:433–41. Edwards J, Mukherjee R, Munro AF, Wells AC, Almushatat A, Bartlett JM. HER2 and COX2 expression in human prostate cancer. Eur J Cancer 2004;40:50–5. Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C, et al. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377–86. Singh T, Vaid M, Katiyar N, Sharma S, Katiyar SK. 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 2011;32:86–92.
VOL. - NO. - / - 2015
Fertility and Sterility® 40.
41.
42.
Fu L, Chen W, Guo W, Wang J, Tian Y, Shi D, et al. 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 2013;8:e69240. Kuo CL, Chi CW, Liu TY. Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells. In Vivo 2005;19:247–52. Liu B, Wang G, Yang J, Pan X, Yang Z, Zang L. Berberine inhibits human hepatoma cell invasion without cytotoxicity in healthy hepatocytes. PLoS One 2011;6:e21416.
VOL. - NO. - / - 2015
43.
44.
45.
Mantena SK, Sharma SD, Katiyar SK. Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol Cancer Ther 2006;5:296–308. Wang L, Liu L, Shi Y, Cao H, Chaturvedi R, Calcutt MW, et al. Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor. PLoS One 2012;7:e36418. Chidambara Murthy KN, Jayaprakasha GK, Patil BS. The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells. Eur J Pharmacol 2012;688:14–21.
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