Objective
Uterine leiomyomas represent a common gynecologic problem with no satisfactory long-term medical treatment. The purpose of this study is to examine the effects of simvastatin on uterine leiomyoma, both in vitro and in vivo.
Study Design
This is a laboratory-based experimental study. For in vitro studies, we used human and rat leiomyoma cells. For in vivo studies, we used immunodeficient mice supplemented with estrogen/progesterone pellets xenografted with human leiomyoma tissue explant.
Results
For in vitro studies, cells were treated with different concentrations of simvastatin for 48 hours. Simvastatin induced dose-dependent apoptosis in leiomyoma cells as measured by a fluorometric caspase-3 activity assay, and inhibited proliferation as demonstrated by an (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (both were significant at 5 and 10 μM). In addition, simvastatin decreased Akt signaling pathway phosphorylation as examined using Western blot analysis. For in vivo studies, animals were treated for 28 days with simvastatin (20 μg/gm body weight/day) vs vehicle control. The treatment inhibited tumor growth as measured weekly using calipers and/ or ultrasound ( P < .01). Finally, simvastatin decreased expression of the proliferation marker Ki67 in xenograft tumor tissue as examined by immunohistochemistry ( P = .02).
Conclusion
Simvastatin can be a promising treatment for uterine leiomyoma. Further studies, including pharmacokinetic and drug delivery studies, are required.
Uterine leiomyomas are the most common tumors of the female genital tract, with 50-70% of women experiencing uterine leiomyomas during their lifetime. They cause several gynecologic symptoms that include heavy bleeding and pelvic pain. Unfortunately, no satisfactory long-term medical treatment is currently available, and ultimately a hysterectomy is needed in many cases. In fact, approximately 600,000 hysterectomies are performed annually in the United States, and approximately one-half of these are related to leiomyomas.
Simvastatin is a semisynthetic member of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor family. Statins have been used to treat hyperlipidemia for >20 years; therefore, they have a well-known safety profile. Remarkably, they were also found to have profound and broad-reaching effects on various types of tissues. Furthermore, statins have cell-specific effects on cellular proliferation. For example, they stimulate proliferation of endothelial progenitor cells, while they inhibit it in vascular smooth muscles, hepatocytes, and certain neoplastic cell lines.
Statins have been shown to have general antitumor properties, particularly against breast cancer and gynecologic malignancies. In addition, recent studies showed that that certain statins, specifically simvastatin and atorvastatin, have beneficial effects on benign steroid-dependent gynecologic conditions, such as endometriosis. Therefore, it seems intriguing to examine the effects of statins on uterine fibroid tumors. We previously demonstrated that simvastatin inhibits proliferation, interrupts cell cycle progression, and induces apoptosis through a calcium-dependent mechanism in human leiomyoma cells in vitro. In this study, we proceeded to investigate the in vivo effects of simvastatin on a patient-derived xenograft leiomyoma murine model. In addition, we investigated the effects of treatment on a human and a rat leiomyoma cell line. We report, for the first time to the best of our knowledge, that simvastatin treatment is associated with tumor growth inhibition in leiomyoma xenograft animal models and possibly has therapeutic potential for the treatment of uterine leiomyoma.
Materials and Methods
Cells
The Eker rat leiomyoma cell line (ELT-3) was a kind gift from Dr Cheryl Walker, professor and director at the Texas A&M Health Science Center Institute of Biosciences and Technology in Houston, TX. These cells were established and have been characterized fully. The immortalized human leiomyoma cell line (huLM) was derived from a patient with a uterine leiomyoma after hysterectomy. They were immortalized by the Dr Darlene Dixon group who used telomerase induction and have been characterized previously.
HuLM cells were cultured and maintained in a Smooth Muscle Growth Medium-2 (SmGM-2; Lonza, Walkersville, MD) that contained 5% fetal bovine serum, 0.1% insulin, 0.2% basic human fibroblast growth factor, 0.1% gentamicin/amphotericin B, and 0.1% human epidermal growth factor; all of which were purchased from Lonza (Walkersville, MD). ELT-3 leiomyoma cells were cultured and maintained in a DF8 medium as previously described. Cells were incubated in a 5% CO 2 atmosphere under 37°C and split once 70-80% confluent.
Materials
The simvastatin was purchased from Cayman Chemical (Ann Arbor, MI). A complete protease inhibitor cocktail without EGTA was purchased from Roche Applied Science (Indianapolis, IN). Z-DEVD-R110 used for the caspase-3 assay was purchased from the American Peptide Company (Sunnyvale, CA). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent was purchased from Calbiochem (EMD Millipore, Merck KGaA, Darmstadt, Germany). Monoclonal antiphospho Akt and anti–total Akt antibodies, which were used for Western blotting, were purchased from Cell Signaling (Danvers, MA). Rabbit polyclonal anti-Ki67 antibody, which was used for immunohistochemistry, was purchased from Abcam (Cambridge, MA).
A Matrigel basement membrane matrix was purchased from BD Biosciences (San Jose, CA). Sixty-day release pellets with 17ß-estradiol (0.05 mg) + progesterone (50 mg) were purchased from Innovative Research of America (Sarasota, FL).
Simvastatin activation
The simvastatin was activated before use to convert the prodrug (lactone) to an active (beta-hydroxyacid) form. Activation was done as previously described. In brief, 25 mg of simvastatin was dissolved in 625 μL of absolute ethanol and then added to 935 μL of 0.1 NaOH. After being mixed, the solution was placed in a water bath at 50°C for 2 hours then diluted with water to the required concentration. Finally, the solution was sterile filtered and kept at 4°C until use.
Proliferation assay
An MTT assay was used to monitor cellular proliferation. ELT-3 cells were seeded into 96-well plates. After 24 hours, the medium was replaced with a medium that contained 0, 0.1, 0.5, 1, 5, and 10 μmol/L of simvastatin. After 48 hours, the MTT assay was performed as previously described.
Caspase-3 assay
Caspase-3 activity was measured with a quantitative fluorometric assay as previously described. In brief, cells were seeded in 10-cm dishes. After 24 hours, the medium was replaced with a medium that contained 0, 0.1, 0.5, 1, 5, and 10 μmol/L of simvastatin. After 48 hours, cells were harvested, and cell lysates were obtained. Cell lysates that contained equal amounts of proteins were loaded in a 96-well plate in addition to a reaction mixture that contained Z-DEVD-R110 (caspase-3 substrate). Caspase-3 activity was measured fluorometrically over a period of 60 minutes.
Tumor samples
Approval from the Institutional Review Board at the University of Texas Medical Branch was obtained, and verbal consents were obtained from patients. Leiomyoma samples (otherwise discarded) from patients who undergo hysterectomies were obtained and immediately transported to the laboratory under sterile conditions. For this study, independent samples were obtained and processed from 2 separate patients at 2 different time points.
Animals
Approval from the Institutional Animal Care and Use Committee at the University of Texas Medical Branch was obtained. All animal handling was performed in accordance with Institutional Animal Care and Use Committee and other appropriate guidelines.
We modified a previously described leiomyoma xenograft animal model. Six-week-old female immunodeficient NOG (NOD/Shi- scid /IL-2Rγ null ) mice were purchased from Taconic (Hudson, NY). These mice have multiple immunodeficiencies and were originally developed by the Central Institute for Experimental Animals in Japan in 2000 from 3 mice strains: NOD/Shi, SCID, and IL-2Rγ null . These combined defects rendered the mice more appropriate as human xenograft models. All procedures were performed under sterile precautions; the mice were kept in an appropriately isolated environment.
Estrogen-progesterone pellet insertion
Mice were anesthetized by isoflurane (1-2%), which was administered by mask. At least 5 days before leiomyoma xenograft placement, 1 17ß-estradiol (0.05 mg)/progesterone (50 mg) 60-day pellet was placed subcutaneously in each animal. For pain control, buprenorphine (0.05–0.1 mg/kg subcutaneously twice daily, then as needed) was administered.
Tissue processing and implantation
Leiomyoma tumors that were obtained from patients were processed immediately under sterile conditions. Uniform 2 × 2 × 3 mm cylinders were obtained from the tumor with the use of a 2-mm Keyes’ biopsy punch and then dipped in the Matrigel basement membrane matrix. The tumors then were inserted subcutaneously into 20 mice through a small skin incision in their flanks, which were then closed with sterile surgical staples (2 tumors per mouse). The animals were observed closely for pain and signs of infection after procedures. One week after xenograft placement, the staples were removed, and treatment was initiated. The 20 animals were assigned randomly to treatment (n = 10) or control (n = 10) groups.
Animal treatment and death
Animals were treated by a daily subcutaneous injection of simvastatin (20 μg/ gm body weight/day) or vehicle control for 28 days. Animals were observed closely on a daily basis, and tumor sizes were measured weekly by caliper. After 28 days, tumor sizes were measured by calipers for the last time.
High-resolution ultrasound system was used to measure tumor sizes in 3 dimensions with the use of an 18-38MHz probe (Vevo 2100; Visualsonics, Toronto, Canada). This measurement was done at the biomedical engineering center at the University of Texas Medical Branch. Tumor volumes were then calculated with the formula volume = 0.52 × length × width × height (this is a simplification of tumor volume = 4/3 × pi × length/2 × width/2 × height/2).
After ultrasound scanning, animal euthanasia was performed with an isofluorane overdose followed by cervical dislocation. Skin incisions were made, and the tumors were removed. Representative images of the tumors by ultrasound scanning and at death are shown in Figure 1 . Tumor sizes were measured again by calipers, and wet tumor weights were obtained. Finally, tumors were placed in a 10% buffered formalin solution and kept at 4°C until immunohistochemistry. The research personnel who administered treatment and measured tumor size were blinded in regard to treatment vs control groups.
Immunohistochemical studies
Tumor tissues were obtained from the animals that were fixed with the use of 10% buffered formalin; blocks were used to prepare tissue sections. These sections were next stained with hematoxylin and eosin to examine the tissue morphologic condition. Further sections were used for immunostaining for the proliferation marker Ki67, with the use of the immunoperoxidase method with DAB as the chromogen. Immunostaining was then quantitated with the use of the Image-Pro Plus software (Media Cybernetics, Rockville, MD). This analysis software generates arbitrary numbers using a computer grading algorithm that takes into account the tissue area, the percentage of positively stained cells to the total number of cells, and the intensity of the staining. This was performed in 10 separate high-power fields (×20) per slide. The pathologist who read the slides was blinded in regard to treatment vs control groups.
Statistical analysis
In vitro experiments were performed in triplicates and independently repeated at least 3 times. Data were checked for normality using the Shapiro-Wilk test. Whenever applicable, data were presented as mean ± SEM (standard error of the mean). For in vitro experiments where several concentrations of simvastatin were used, 1-way analysis of variance (ANOVA) was used with Bonferroni post-hoc analysis to analyze each treatment concentration vs control. Different statistical methods were used for the in vivo experiments. To evaluate within-animal concordance of the volumes of the 2 xenografts in each animal, we used intraclass correlation coefficient. This was calculated with SPSS software for Windows (version 20; IBM Corporation, Armonk, NY). We used the Student t test to compare tumor size measurements and immunohistochemistry results between the treatment and control groups. A 2-sided alternative to the null hypothesis of no difference was used. Probability values < .05 were considered statistically significant. We used SigmaPlot software (Systat Software Inc, San Jose, CA) for the statistical analysis.
Results
Induction of apoptosis in leiomyoma cells in vitro
To investigate the effects of simvastatin on apoptosis, we treated ELT-3 cells with different concentrations of simvastatin for 48 hours, followed by a fluorometric caspase-3 activity assay with cell lysates. ANOVA analysis demonstrated significant difference among groups ( P = .004). These results are similar to our previous findings using HuLM cells. Bonferroni post-hoc analysis showed significant differences at 5 and 10 μmol/L compared with controls ( P = .005 and .025, respectively; Figure 2 ).
Inhibition of proliferation of leiomyoma cells in vitro
Next, we proceeded to determine whether simvastatin affects proliferation of leiomyoma cells. After treating ELT-3 cells for 48 hours, we performed an MTT assay. ANOVA analysis demonstrated significant difference among groups ( P = .019). These results are similar to our previous findings with HuLM cells. Bonferroni post-hoc analysis showed that simvastatin significantly inhibits proliferation of cells at 10 μmol/L ( P = .021; Figure 3 ).
Inhibition of Akt signaling pathway in leiomyoma cells in vitro
We next sought to examine the effects of simvastatin on proliferation signaling pathways. To this end, we used Western blotting to examine the phosphorylation of Akt protein in HuLM cells. Phosphorylation was calculated by dividing the expression of phosphorylated fraction by the total Akt protein. ANOVA demonstrated a statistically significant decrease in Akt phosphorylation ( P = .002), with post-hoc analysis showing the 2, 5, and 10 μmol/L concentrations to be statistically significant ( P = .049, .017, and .014, respectively; Figure 4 ).
Inhibition of leiomyoma xenograft growth in the animal model
To determine the antitumor efficacy of simvastatin in a patient-derived xenograft leiomyoma model, mice were treated with a daily dose of 20 μg/kg simvastatin (treatment group; n = 10) or vehicle control (control group; n = 10). Animals in both groups were checked regularly for any signs of poor general health, and both groups stayed healthy until death. Because each animal received 2 tumor xenografts, we had 20 tumors in each group (total of 40 tumors). The sizes of tumors were measured weekly by calipers. At the time of death, the tumor sizes were measured by both calipers and ultrasound scanning. The intraclass correlation coefficient (a concordance measure) of the volumes of the 2 xenografts in each animal was 0.576. As graphically presented in Figure 5 , tumor sizes were 37.9% and 49.1% smaller in the treatment group compared with controls at weeks 3 and 4 ( P = .003 and .002, respectively). In addition, as measured by ultrasound scanning before death, the mean tumor size was 43.1% smaller in the treated group compared with the control group ( P = .043; Figure 5 , C). Finally, the mean wet tumor weight was 28.5% smaller in the treated group compared with the control; however, this difference did not reach significance ( P = .17; Figure 5 , D).
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