Berberine Inhibits Uterine Leiomyoma Cell Proliferation via Downregulation of Cyclooxygenase 2 and Pituitary Tumor-Transforming Gene 1

Abstract

We previously demonstrated that berberine (BBR) inhibits cell proliferation and induces apoptosis in a human uterine leiomyoma (UtLM) cell line but does not demonstrate a significant cytotoxic effect in a normal human uterine smooth muscle (UtSM) cell line. However, the mechanisms of this inhibition are unclear. Of note, cyclooxygenase 2 (COX2) and pituitary tumor-transforming gene 1 (PTTG1) are overexpressed in human uterine leiomyomata and are involved in the pathogenesis of uterine fibroids (UFs). We found that COX2 and PTTG1 were overexpressed in UtLM and that BBR decreased COX2 and PTTG1 expression in UtLM cells. Our data support that UtLM and UtSM are immortalized cell lines without phenotypic alterations from parental cell types and suggest that COX2 and PTTG1 are molecular targets for BBR. However, studies in these cell lines may not reveal all activities of BBR in vivo, and we therefore proceeded to test in this report the antitumor effects of BBR in an UF nude mouse xenograft model. When UF nude mice were killed at 7 weeks, tumor weight in controls was 45 ± 7 mg versus 20 ± 3 mg (P < .05) in the low-dose (5 mg/kg) and 7 ± 3 mg (P < .01) in the high-dose (10 mg/kg) BBR groups, respectively. Expression of proliferation markers, cell cycle–related genes, and UF-related genes was downregulated in tumors. No unusual behavioral changes and no signs of kidney or liver damage were observed in the animals with BBR treatment. In conclusion, our data suggests that (a) COX2 and PTTG1 are molecular targets for BBR and (b) BBR is potentially an effective and safe anti-UF agent.

Keywords

uterine fibroids leiomyoma berberine COX2 PTTG1

Introduction

Uterine fibroids (UFs), also called uterine leiomyoma (UtLM), are the most common benign smooth muscle tumors in reproductive-age women. The UFs affect over 50% of women aged 35 to 49 years, with an even higher incidence of 70% to 80% among African American women. Symptoms of UFs include pelvic pain, vaginal bleeding, anemia, miscarriage, and infertility.^1,2 The economic burden of UFs in the United States ranges from US$5.9 to US$34.4 billion/year.³ Current treatment options include traditional and minimally invasive surgery and medicinal agents. Surgery, whether open or endoscopic, is costly and associated with operative morbidity and mortality. Medicinal agents, such as long-acting gonadotropin-releasing hormone (GnRH) analogs, generally result in significant hypoestrogenism,⁴ thereby limiting their utility as a long-term treatment option. Hence, there is a pressing need to identify long-term medical treatment options for UFs that can be effective with minimal side effects.

Berberine (BBR) is a chemical compound extracted from several plants and a common and cost-effective remedy that has been safely used for centuries in North American folk and traditional Chinese medicines.⁵ Berberine has shown efficacy against a variety of human cancers such as melanoma, lung cancer, neuroblastoma, colonic carcinoma, breast cancer, and hepatocellular carcinoma.⁵ These effects have been observed in both in vitro and in vivo studies, as assessed by suppression of tumor cell proliferation, induction of tumor cell apoptosis, and inhibition of tumor invasion and metastasis.⁶ In a previous report, we demonstrated that BBR inhibits spontaneous and estrogen- or progesterone-induced immortalized UtLM cell proliferation, and it induces apoptosis in UtLM in clinically relevant concentrations but does not demonstrate significant cytotoxic effect in human uterine smooth muscle (UtSM) cell lines.⁷ The mechanisms underlying these effects remain unclear. Cyclooxygenase 2 (COX2) and pituitary tumor-transforming gene 1 (PTTG1) are overexpressed in human UtLM cells and involved in the pathogenesis of UFs.^8
–10 Berberine is a natural COX2 inhibitor and inhibits cell growth in human non–small cell lung cancer cells.¹¹ We tested COX2 and PTTG1 as molecular targets for BBR in this report.

In addition, our previous in vitro results suggest BBR as a potential therapeutic agent for the medical treatment of UFs.⁷ However, although the 2 transformed cell lines previously used, UtLM and UtSM, originally arise from humans and demonstrate no phenotypic alteration from the parental cell types,¹² studies in these cell lines may not reveal all activities of BBR in vivo. Furthermore, the concentrations of BBR in cell culture may not reflect the actual concentration of BBR experienced by tissues in vivo with therapeutic administration. Therefore, in the present study, we tested the in vivo antitumor effects of BBR on UF development in a nude mouse tumor xenograft model. We also examined the safety of BBR treatment in these mice by monitoring liver and kidney function.

Materials and Methods

Cell Culture

Immortalized UtLM and UtSM cell lines were originally generated by Dr Darlene Dixon via transfection with human telomerase gene without phenotypic alteration from parental cell types.¹² Cells were maintained in Dulbecco modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). For the BBR stimulation experiments, BBR (cat# B3251, purity ≥ 98%; Sigma-Aldrich, St. Louis, Missouri) was directly added to maintenance medium. Eker rat uterine leiomyoma (ELT3) cells were maintained in DMEM containing 10% FBS. All experiments were performed in triplicate.

Cell Proliferation 3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium Assay

Cell proliferation was determined using the CellTiter 96 Cell Proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay kit (Promega, Madison, Wisconsin). Experiments were conducted in 96-well plate with 5000 cells/well initially. After treatment for 72 hours, cells were washed twice with phosphate-buffered saline (PBS) and incubated in 100 µL per well of DMEM. Twenty microliters of CellTiter 96 solution was added to each well. Absorbance was determined with a microplate reader at 490 nm.

Transfection of Small Interfering RNA and Plasmid

Six-well and 96-well plates of UtLM and UtSM cells were used for transfection by following the manufacturer’s instructions. Lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, California) was used to transfect small interfering RNAs (siRNAs), and MegaTran 1.0 (OriGene, Rockville, Maryland) was used to transfect plasmids. Plasmids were purchased from OriGene (Rockville, Maryland), and siRNAs were purchased from Life Technologies (Carlsbad, California). Gene expression was determined at 48 hours posttransfection, and cell proliferation was investigated at 72 hours posttransfection.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was extracted using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher, Waltham, Massachusetts). First strand complementary DNA (cDNA) synthesis was achieved using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California). Real-time quantitative polymerase chain reaction (PCR) was performed using an iTag Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc, Hercules, California) on an Applied Biosystems 7300 Real-time PCR System. Human primers for COX2 and PTTG1 as well as rat primers for 2 cell proliferation markers (Mki67 ¹³ and proliferating cell nuclear antigen [PCNA]¹⁴), 2 G2/M phase-related genes (Cyclin B1, Cyclin-dependent kinase 1 [Cdk1])^15
–17 and 2 genes that are typically overexpressed and play important roles in the pathogenesis of UtLMs (PTTG1 and COX2)^8,10,18 were purchased from www.realtimeprimers.com. ACTB for human and Actb for rat were used as internal controls. Relative fold change of target gene expression was calculated using the 2^−ΔΔCt method.

Western Blots

Forty micrograms of total protein were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were immunoblotted with the appropriate primary antibodies at 4°C overnight. Antibodies for PTTG1, Mki67, and PCNA were purchased from Santa Cruz Biotechnology (Dallas, Texas), and COX2 was purchased from Cayman Chemical (Ann Arbor, Michigan). After washing, membranes were incubated with a secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, Pennsylvania), detected with chemiluminescence reagent (Thermo Scientific, Hampton, New Hampshire), and exposed by autoradiography.

Tumor Induction and BBR Treatment in Nude Mouse Xenograft Model

Fifteen 5- to 6-week-old female athymic nude mice (Foxn1^nu) were purchased from Jackson Laboratory (Bar Harbor, Maine). One 60-day estrogen pellet (17β-estradiol 1.7 mg; Innovative Research of America, Sarasota, Florida) was surgically implanted under the neck skin of the female nude mice. Three days postimplantation, mice were inoculated subcutaneously via the right flank region with 1 × 10⁷ ELT3 cells in 300 μL serum-free media. Mice were randomized into 3 groups (5 per group): (1) controls (no treatment), (2) low-dose BBR (5 mg/kg), and (3) high-dose BBR (10 mg/kg). We chose these 2 doses because prior studies demonstrated in a lung cancer xenograft model that BBR treatment at these concentrations decreased tumor size by 20% and 80%, respectively.¹⁹

Mice in the low- and high-dose groups were intraperitoneally injected with BBR in PBS every other day, whereas control mice received an equal volume of PBS only. Tumor size(s) was measured with calipers in 3 dimensions with the formula: 0.52 × length × width × height²⁰ twice a week. Animals were euthanized 7 weeks post injection. All animal experiments were approved by the Institutional Animal Care and Use Committee at Augusta University.

Serum Aspartate Transaminase, Alanine Transaminase, and Creatinine Measurement

The level of the hepatic function indices, aspartate transaminase (AST) and alanine transaminase (ALT), and renal function index, creatinine, were measured in serum by the Yale Mouse Phenotypic Center (New Haven, Connecticut).

Statistical Analysis

Comparisons of 2 groups were carried out using the unpaired t test (XLSTAT Software, New York). Comparisons of multiple groups were carried out by analysis of variance followed by a posttest using the Tukey (among groups) and Dunnett (compared to the control group) tests (XLSTAT Software, New York). Data are presented as mean ± standard error (SE). Significant differences were defined as P < .05.

Results

Cyclooxygenase 2 and PTTG1 Are Overexpressed in UtLM

The COX2 and PTTG1 were significantly overexpressed in UtLM compared to UtSM at both messenger RNA (mRNA; Figure 1A and B) and protein levels (Figure 1C and D). For COX2, the mRNA and protein levels in UtSM were nearly undetectable (Figure 1A and C).

Cyclooxygenase 2 and PTTG1 Regulate Cell Proliferation in UtLM and UtSM Cells

Reduced expression of COX2 or PTTG1 by siRNA significantly decreased cell proliferation in UtLM (Figure 2A and B and Supplemental Figure 1A and 1B) but had no effect in UtSM cells (Figure 2A–C). However, overexpression of COX2 or PTTG1 by plasmid transfection stimulated UtSM cell proliferation (Figure 2D and Supplement Figure 1C and D).

Berberine Inhibits COX2 and PTTG1 Expression in UtLM but not in UtSM Cells

Berberine treatment did not alter the expression of COX2 (Figures 3A [mRNA] to 3C [protein]) or PTTG1 (Figures 3E [mRNA] to 3G [protein]) in UtSM Cells. However, BBR treatment significantly reduced the expression of COX2 (Figures 3B [mRNA] to 3D [protein]) and PTTG1 (Figures 3F [mRNA] to 3H [protein]) in UtLM cells in a dose-dependent manner.

Berberine Inhibits ELT3 Cell Proliferation In Vitro

Berberine treatment significantly reduced ELT3 cell viability in a dose-dependent manner (Figure 4A) and inhibited ELT3 cell growth by approximately 40% and 60% at concentrations of 50 and 100 µM, respectively.

Berberine Inhibits ELT3 Tumor Development In Vivo in Nude Mouse Xenograft Model

All animals developed visible tumors after inoculation with ELT3 cells but appeared otherwise healthy. Figure 4B depicts representative mice with tumors in the control (Figure 4B-1 and B-4), low-dose (Figure 4B-2 and B-5), and high-dose (Figure 4B-3 and B-6) BBR treatment groups at 7 weeks posttreatment. The progression of tumor size slowed in BBR-treated mice. At 7 weeks posttreatment, tumor volume reached 50.7 ± 4.2 mm³ in control animals. However, in BBR-treated mice, tumors were significantly smaller, 11.6 ± 3.1 mm³ (P < .05 vs control) in the low-dose group and 4.5 ± 2.3 mm³ (P < .01 vs control) in the high-dose group, respectively (Figure 4C). One mouse in the BBR 10 mg/kg treated group demonstrated no clinically evident tumors at the end of the seventh week of treatment. The difference in tumor weight was consistent with tumor size when the animals were killed at 7 weeks. Tumor weight in controls was 45.0 ± 6.9 mg versus 20.0 ± 3.0 mg (P < .05) in the low-dose and 6.7 ± 2.5 g (P < .01) in the high-dose BBR-treated mice, respectively (Figure 4D).

Berberine Downregulates Proliferation, Cell Cycle, and Fibroid Pathogenesis-Related Genes in ELT3 Cell-Induced Tumors

The mRNA and protein expression of proliferation markers (Mki67 and PCNA), cell cycle-related genes (Cyclin B1 and Cdk1), and UF pathogenesis-related genes (COX2 and PTTG1) was observed to be significantly downregulated by BBR treatment in the ELT3 cell-induced tumors (Figure 4E and F).

Safety Assessment

No unusual behavioral changes were observed, and all mice gained weight (Figure 5A) during the 7-week experiment. Final weights of the body, liver, and kidney were not different among treatment groups (Table 1). In serum, the concentrations of AST, ALT, and creatinine were also not different between groups (Figure 5B).

Table 1.

Final Weight After 7 Weeks of Study (n = 5 per group).

Final Weight, Mean± SD, g	Control	Low-Dose BBR (5 mg/kg)	High-Dose BBR (10 mg/kg)
Body	24.38 ± 2.10	25.51 ± 1.53	24.66 ± 1.22
Liver	1.51 ± 0.12	1.56 ± 0.27	1.50 ± 0.13
Kidney	0.41 ± 0.03	0.44 ± 0.08	0.44 ± 0.02

Abbreviation: BBR, Berberine; SD, standard deviation.

Discussion

The COX2 and PTTG1 are overexpressed in human UtLMs compared to match healthy myometrial tissue.^8
–10 The inhibition of COX2 activity by COX2 inhibitors celecoxib or NS-398 significantly reduced the proliferation of leiomyoma cells, whereas overexpression of PTTG1 significantly induced the proliferation of leiomyoma cells. These results suggest that COX2 and PTTG1 play an important role in the pathogenesis of UFs.^8
–10 These 2 pathogenesis-related genes were tested in the immortalized cell lines UtLM and UtSM. Similar to the primary cells, COX2 and PTTG1 were also overexpressed in UtLM compared to UtSM. In addition, COX2 or PTTG1 knockdown inhibited UtLM cell proliferation, whereas COX2 or PTTG1 overexpression increased UtSM cell proliferation. Our data support the concept that UtLM and UtSM are immortalized cell lines without phenotypic alteration from parental cell types.¹² These cells could be used in studying the molecular pathways involved in the transformation of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of these tumors.

We previously reported that BBR inhibits UtLM cell proliferation at low but clinically relevant concentrations, and treatment did not demonstrate a cytotoxic effect on UtSM cells.⁷ However, the mechanisms of BBR’s antifibroid effects are not clear. In the present study, we found that BBR inhibited COX2 and PTTG1 expression in a dose-dependent manner in UtLM but not in UtSM cells. These in vitro data strongly suggests that COX2 and PTTG1 are the molecular targets of BBR in UFs.

Our previous in vitro study suggests that BBR may be a potential therapeutic agent for the medical treatment of UtLMs. To investigate BBR’s effect in vivo, we tested BBR in tumors induced in nude mice using a rat ELT3 cell line. In this animal model, we chose 2 doses of BBR (5 and 10 mg/kg), which had been previously demonstrated to decrease tumor size by 20% and 80% in a murine lung cancer xenograft model.¹⁹ With injections every other day, these 2 doses of BBR were able to decrease tumor weight by 60% and 85%, respectively. In the ELT3 cell-induced tumors, BBR downregulated mRNA and protein expression of proliferation markers (Mki67 and PCNA), cell cycle-related genes (Cyclin B1 and Cdk1), and UF pathogenesis-related genes (COX2 and PTTG1) in a dose-dependent manner. That BBR downregulated the expression of COX2 and PTTG1 in these tumors further supports the concept that COX2 and PTTG1 are 2 molecular targets for BBR in UFs. Regarding safety, BBR administration to our mice did not result in any significant negative impact on weight, behavior, or hepatic or renal indices.

Berberine has been found to preferentially inhibit the estradiol 4-hydroxylation activity of cytochrome P450 1B1 variants, suggesting that 4-hydroxyestradiol-mediated toxicity might be reduced by BBR.²¹ In addition, our data indicated that BBR alone was able to inhibit ELT3 cell proliferation in vitro (Figure 4A), and BBR inhibited both spontaneous and estradiol-stimulated cell proliferation in UtLM cells.⁷ These data suggest that in addition to direct inhibition of the growth of ELT3 cells, BBR may also alter metabolism of estradiol used to support the growth of fibroid cells and thus indirectly inhibiting ELT3 tumor formation in the mouse model.

In addition to BBR, there are other Chinese herbal preparations that have shown efficacy in suppressing UFs including Tripterygium wilfordii, Guizhi Fuling formula, Gongliuqing capsule, Lenge Xiaozheng Tang, Jiliu Tang, and green tea extract (epigallocatechin gallate).^22
–24 These herbal medicines have demonstrated beneficial effects in reducing the volume of the uterus and UFs in clinical trials. However, current evidence does not support or refute the use of herbal preparations for the treatment of UFs due to insufficient number of studies with adequately large sample sizes and of sufficient high quality.^22,23 Furthermore, high-quality clinical trials evaluating clinically relevant outcomes are warranted.²²

In conclusion, our in vitro and in vivo data suggest that (1) COX2 and PTTG1 are molecular targets of BBR and (2) BBR is potentially an effective and safe anti-UF agent. However, as the ELT3 cells used to induce tumors in our nude mouse model are derived from Eker rat uterine tumors, which may differ from human UFs cells,²⁵ we will extend our BBR studies to a patient-derived xenograft model for UF.

Footnotes

Authors’ Note

The reported work was done at Augusta University. Tung-Yueh Chuang and Jie Min have contributed equally to this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Augusta University research funds (to Y.H.C).

Supplemental Material

Supplementary material is available for this article online.

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