Human Leiomyoma Research Articles

Mifepristone, a clinically effective leiomyoma therapy, directly regulates fibrosis in human 3-dimensional cultures

Treatment of human leiomyomas with mifepristone results in decreases in leiomyoma size and symptoms, but the mechanism remains unclear. Our goal was to characterize the impact of mifepristone on extracellular matrix (ECM) formation in human 2-dimensional (2D) and 3-dimensional (3D) leiomyoma cultures.

Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of fibroids

Leiomyomas, commonly known as fibroids, are the most common smooth muscle overgrowth in women and a major indication for hysterectomy. The molecular mechanisms of leiomyomas remain largely unknown. Leiomyomas show increased cell proliferation as compared to myometrium. The use of antioxidant strategies is thought to be beneficial in treatment of fibroids. The objective of this study is to determine the differential expression levels of superoxide dismutase (SOD3) and catalase (CAT), as well as the effect of hypoxia, in human leiomyoma versus myometrium cells.

Gonadotropin-releasing hormone (GnRH) agonist leuprolide acetate and GnRH antagonist cetrorelix acetate directly inhibit leiomyoma extracellular matrix production

To determine the direct effect that GnRH analogues leuprolide acetate and cetrorelix acetate have on extracellular matrix in human leiomyoma and patient-matched myometrial cells. Laboratory study. University hospital. None. Cell culture, proliferation studies, and messenger RNA and protein analysis. Expression of GnRHR1, COL1A1, fibronectin, and versican variant V0 in treated leiomyoma cells and patient-matched myometrial cells. Leiomyoma cells were treated with GnRH analogues for 6, 24, and 120 hours. Leuprolide treatment for 6 hours resulted in an increase in expression of GnRHR1 (4.02 ± 0.12-fold), COL1A1 (6.41 ± 0.29-fold), fibronectin (9.69 ± 0.18-fold), and versican variant V0 (7.58 ± 0.43-fold). Leiomyoma cells treated with cetrorelix for 6 hours showed a decreased expression of GnRHR1 (0.5 ± 0.15-fold), COL1A1 (3.79 ± 0.7-fold), fibronectin (0.92 ± 0.09-fold), and versican variant V0 (0.14 ± 0.07-fold). Leuprolide treatment of leiomyoma cells at high concentrations (10(-5) M) did not result in an increase in protein production. Cetrorelix treatment of leiomyoma cells for 6 hours showed an increase in fibronectin protein production (3.14 ± 0.09-fold). Protein production of leiomyoma cells treated with cetrorelix for 120 hours demonstrated a decrease in GnRHR1 (0.51 ± 0.07-fold), COL1A1 (0.35 ± 0.07-fold), fibronectin (1.94 ± 0.08-fold), and versican variant V0 (0.77 ± 0.19-fold). Our findings demonstrate that GnRH analogue treatment directly regulated COL1A1, fibronectin, and matrix proteoglycan production. The reduction in versican variant V0 gene expression caused by cetrorelix treatment, and its association with the osmotic regulation of leiomyomas, presents a new and innovative approach to therapy for this disease.

Down-Regulation of miR-29b Is Essential for the Pathogenesis of Uterine Fibroids.

Uterine leiomyomas (ULs) are the most common neoplasm in women of reproductive age with an accumulative incidence of over 70%. They are believed to arise from the myometrium, but the etiology of this tumor remains unknown. The most notable characteristic of UL is the excess extracellular matrix (ECM), composed mainly of collagen Types I and III. It has been repeatedly reported that many collagen genes (COL1A1, 1A2, 3A1, 5A1, 5A2, and 7A1) are up-regulated in ULs. Since the production of ECM protein is one of the factors that contribute to the tumor growth, it is important to understand the mechanism of collagen over-production in ULs. In addition to collagens, mRNAs of many genes are differentially expressed in the myometrium and ULs. Recent gene expression profiling studies have revealed that many microRNAs (miRNAs) are also aberrantly expressed in ULs. Specifically, miR-29b is consistently found to be down-regulated in ULs in comparison to normal myometrium. Down-regulation of miR-29 has been correlated with an over-expression of collagen in the liver, lung, kidney and cardiac fibrosis. In vitro studies have demonstrated that miR-29b can down-regulate multiple collagen genes including COL1A1, 1A2, 5A1, and 5A2. Moreover, COL3A1 was ranked top among ~1000 potential targets of miR-29b in a target prediction algorism (miSVR) based on the Support Vector Regression model. These observations suggest that the down-regulation of miR-29b may play a role in the overproduction of ECM in ULs. To investigate the role of miR-29b in the pathogenesis of UL, we first established stable human leiomyoma cell lines that restored miR-29b expression levels to normal by lentiviral infection. The miR-29b and control cell lines were then compared in cell proliferation and the expression of potential miR-29b targets including COL1A1 and 3A1. While the growth rate was not affected by an up-regulation of miR-29b, genes for ECM protein including the collagens were significantly down-regulated. It is well known that myometrial and UL cells lose their original characteristics in vitro. Therefore, we further examined the effect of miR-29b expression levels on tumor growth in vivo by utilizing the xenograft model. When the miR-29b level was restored to the level of myometrium, UL cells did not form a solid tumor. Instead, the UL cells grew flat on the surface of the host mouse kidney. Histological analysis indicated that an up-regulation of miR-29b inhibited collagen accumulation. The experiment was repeated with primary human UL cells, and it confirmed the result of the cell line experiment. Thus, the over-production of ECM in UL is due to the down-regulation of miR-29b, which targets mRNAs of multiple collagens including two major collagen genes, COL1A1 and 3A1. Reversely, we generated myometrial cell lines in which miR-29b was permanently knocked down. While the knock-down of miR-29b increased the levels of collagen in vitro, the down-regulation of miR-29b itself was not sufficient to make myometrial cells tumorigenic in vivo. When cell lines were grafted into mice, neither the mir-29b knock-down nor the control cell lines formed tumors. In conclusion, down-regulation of miR-29b is essential for the formation of ULs, but it is not sufficient to transform myometrium into ULs. Administration of miR-29 mimics may prove to be an effective treatment for symptomatic ULs by reducing the tumor volume. This research was supported by NIH/NICHD Grant R01 HD064402.

Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells

To isolate and characterize human leiomyoma stem cells by the side population (SP) method. Prospective experimental human and animal study. University research laboratory-affiliated infertility clinic. Women undergoing laparoscopic myomectomy. Female non-obese diabetic severe combined immune deficiency (NOD-SCID) mutation mice. Obtainment of human leiomyoma SP cells as candidate tumor-initiating cells and establishment of two leiomyoma SP lines. Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cDNA microarrays hybridization, cell culture, karyotype, molecular analysis, immunocytochemistry, invitro differentiation, xenotransplantation assays, immunohistochemistry. SP cells from human leiomyomas were isolated, identified, and characterized. Two leiomyoma's SP cell lines with a normal karyotype were thus established. Undifferentiated status was confirmed by the expression of OCT-4, NANOG, DNMT3B, GDF3. Presence of typical mesenchymal markers (CD90, CD105, CD73) and absence of hematopoietic stem cell markers (CD34, CD45) supported their mesodermal origin. Mesenchymal lineage commitment was also demonstrated by their ability to differentiate invitro into adipogenic and osteogenic lineages. Finally, their functional capability was established in an animal model by leiomyoma tissue reconstruction. SP cells from human leiomyoma exhibit key features of tumor-initiating cells, opening up new possibilities of understanding the origin and developing new nonsurgical approaches for leiomyomas.

Abstract 1357: Creation and establishment of xenograft model for uterine leiomyoma in immunodeficient mice

Abstract Objective: Uterine leiomyomas are the most prevalent benign tumors in women of reproductive age and the leading indication for hysterectomy in premenopausal women. The underlying causes of uterine leiomyomas are poorly understood, as a result, in part, of the absence of a good model system with which to study these tumors. To overcome this problem, this study was undertaken to develop a murine model for human leiomyoma. Study design: Human leiomyoma tissues were cut into small pieces and some of those were cultured. Tissue pieces and cultured cells were inserted subcutaneously in the flank of each severe immunodeficiency (NOD/SCID gamma, NSG) mice (n=6). Estrogen supplement was needed to maintain serum estrogen level 3 days before the tissue or cultured cells transplantation. Xenograft tissues or cells were harvested after 6wks and analyzed. Results: Six uterine leiomyoma xenografts were successfully established with the tumor tissue derived from six patients. The xenografted human uterine leiomyoma tissues retained their gross appearance with small vessels around the implant tissues and did not change in size up to 6wks after implantation. H&E staining showed that xenografts retained major histologic features of the original leiomyoma. The immunohistochemical staining of SMA(smooth muscle actin) revealed strong cytoplasmic and membranous positivity of spindle tumor cells of xenografts.Conclusions: In summary, transplantation of surgical specimens from uterine leiomyoma patients into NSG mice results in an attractive model for the study of the uterine leiomyoma and may also be useful for analysis or new experimental therapeutic approaches for the treatment of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1357. doi:1538-7445.AM2012-1357

A potential diagnostic biomarker: Proteasome LMP2/b1i-differential expression in human uterus neoplasm

AbstractUterine leiomyosarcoma (ULMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine ULMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker which distinguishes malignant ULMS from benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine ULMS, to establish a treatment method. Proteasome low-molecular mass polypeptide 2(LMP2)/b1i-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. We found LMP2/b1i expression to be absent in human LMS, but present in human LMA. Therefore, defective-LMP2/b1i expression may be one of the risk factors for ULMS. LMP2/b1i is a potential diagnostic-biomarker for uterine ULMS, and may be a targeted-molecule for a new therapeutic approach.

Relationship Between Mast Cell Subtypes and Cyclooxygenase-2 Expression in Uterine Leiomyomas

Objective: Mast cells (MCs) have been proved to be multifunctional cells which are known to be located at peri-and intra-tumoral sites, playing an active role physiologically or pathologically. In humans, MCs are subtyped by the difference in their neutral protease content. MCs that have granules containing tryptase alone (MCT) are predominantly found at mucosal sites. MCs whose granules possess tryptase along with chymase (MCTC) are found especially within connective tissue. MC mediators have different functions and the coexistence of both MC subtypes contributes to tissue homeostasis. Furthermore, cyclooxygenase-2 (COX-2) is an enzyme associated with inflammation, cell growth and differentiation, prevention of apoptosis and tumorigenesis. Recent studies show the clinical significance of COX-2 expression in different tumor types. However, to our knowledge, there is no data assessing the relationship between COX-2 and MC density in uterine leiomyomas. We aimed to investigate the relation between the expression of COX-2, MC density, and proportional changes of MC phenotypes in human leiomyoma uteri and control cases, and also their possible correlations between each other. Methods: We performed a retrospective study of 34 cases carried out on parafin-embedded samples obtained from 14 control (group 1) and 20 leiomyoma uteri (group 2) patients who had undergone curative hysterectomy. These specimens were investigated immunohistochemically by using anti-cyclooxygenase-2 (COX-2) antibodies to stain COX-2; anti-tryptase antibodies to stain MCT and anti-chymase antibodies to stain MCTC. Results: In leiomyoma, the median value of COX-2 staining grade was 2 (min:1-max:3) which was statistically lower than that of controls with a median value of 3 (min:2-max:4) (p=0.001). The median value of tryptase expression was 12 (min:10-max:16) in the control myometrium, slightly higher than that of the leiomyoma group which had a median value of 10.5 (min:6-max:15). On the other hand, the median value of chymase expression was 7 (min:4-max:13) in the control myometrium, which was significantly higher (p=0.001) than that of the leiomyoma group with a median value of 5 (min:2max:8). Thus, in the leiomyoma group, the mast cell subtypes, including tryptase÷chymase proportion, was found to be significantly higher than the controls (p=0.032). Moreover, COX-2, tryptase and chymase expressions showed no correlation in both groups. Conclusion: This study has demonstrated that expressions of COX-2 and mast cell subtypes are reduced in leiomyoma, and the proportion of mast cell subtypes of MCT÷MCTC increases in leiomyoma compared to the control group. However, MC subtypes neither correlated with each other nor with COX-2 expression in the leiomyoma and control series. Understanding the mechanisms for MC functions and the secretory molecules will provide a basis for a rational approach to the development of antitumoral therapy in patients with leiomyoma and other tumors. (JAREM 2011; 1: 44-8)

Recombinant H2 relaxin inhibits apoptosis and induces cell proliferation in cultured leiomyoma cells without affecting those in cultured normal myometrial cells

To evaluate the effects of human relaxin on proliferation and apoptosis in cultured human uterine leiomyoma cells and normal myometrial cells. In vitro experiment. Research laboratory at Kobe University Graduate School of Medicine. Nine patients undergoing hysterectomy for uterine leiomyoma. Cultured leiomyoma cells and normal myometrial cells were treated with human recombinant (rH2) relaxin. Human relaxin receptor LGR7 expressions in cultured leiomyoma cells and myometrial cells were evaluated by immunocytochemical staining. Cell proliferation, proliferating cell nuclear antigen-positive rate, and TUNEL-positive rate were assessed by MTS assay, immunocytochemistry, and TUNEL assay, respectively. Caspase-3 expression was evaluated by Western blot analysis. LGR7 expression was observed both in cultured human leiomyoma cells and myometrial cells. Compared with untreated control cultures, treatment with rH2 relaxin increased the number of viable cultured leiomyoma cells and the proliferating cell nuclear antigen-positive rate in those cells but not in myometrial cells. Moreover, treatment with rH2 relaxin decreased the TUNEL-positive rate in cultured leiomyoma cells but not in myometrial cells. Similarly, Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 in cultured leiomyoma cells but not in myometrial cells. These results suggest that rH2 relaxin selectively inhibits apoptosis by down-regulating caspse-3 expression and induces proliferation in cultured human leiomyoma cells without affecting apoptosis or proliferation in normal myometrial cells.

Adipocytes Enhance the Proliferation of Human Leiomyoma Cells Via TNF-α Proinflammatory Cytokine

Obesity is a well-documented risk factor for uterine leiomyoma with a major impact on women health and health care system of the nation. Obesity is associated with increased secretion of adipokines that significantly influence growth and proliferation of tumor stroma and malignant cells. Adipokines, such as tumor necrosis factor α (TNF-α), are produced in the adipose tissue with concomitant expression in other organs and tissues. Increased and sustained cytokine production is associated with alterations in cell growth and differentiation. We, therefore, explored the influence of human adipocytes (SW872 cells)-mediated biological humoral factors on human uterine leiomyoma (HuLM) cells. We measured cell proliferation and expression of cell-proliferating proteins (proliferating cell nuclear antigen [PCNA], cyclin D1, and B-cell lymphoma 2 [BCL-2]) in human leiomyoma cells cocultured with SW872 cells. SW872-conditioned media was neutralized for TNF-α and proliferation of HuLM cells was observed along with antiapoptotic marker, BCL-2, using Western immunoblot. We found that both SW872-conditioned media and coculture with SW872 cells increased HuLM cell proliferation significantly (P < .05). We determined that this effect was associated with the upregulation of specific markers for proliferation, such as PCNA, cyclin D1, and BCL-2 (P < .05). Furthermore, the addition of neutralizing antibodies, anti-TNF-α, to SW872-conditioned media reversed the proliferation of leiomyoma cells and induced apoptosis as indicated by the reduced expression of antiapoptotic marker BCL-2. SW872 cells secrete TNF-α, which is associated with a proliferative gene profile in HuLM cells and may play a role in initiation and/or progression of uterine leiomyoma.

Simvastatin Induces Apoptosis and Inhibits Proliferation in Human Leiomyoma Cells

Simvastatin Induces Apoptosis and Inhibits Proliferation in Human Leiomyoma Cells

Involvement of proteasome b1i subunit, LMP2, on uterine leiomyosarcma tumorigenesis

AbstractAlthough the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma (LMS) is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone; however, the development of human uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine LMS from benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with human uterine LMS, in order to establish a clinical treatment method. Homozygous deficient mice for a proteasome subunit b1i/LMP2 spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine LMS was demonstrated, but present in human LMA. Therefore, loss in LMP2 expression may be one of the risk factors for human uterine LMS. LMP2 may be a potential diagnostic-biomarker and targeted- molecule for a new therapeutic approach.

Leiomyoma growth mediated by disruptions in injury response and angiogenesis

Thrombospondin 1 (TSP1) is an extracellular matrix component that interacts with proteoglycans, integrins, fibrinogen, and fibronectin. It is involved in cell proliferation, apoptosis, angiogenesis inhibition, and activates latent transforming growth factor beta 1. TSP1 is secreted in response to injury, and high expression is restricted to sites of tissue remodeling or injury. Given dependence of leiomyoma formation on both TGF-beta mediated aberrant injury response and angiogenesis, we investigated the role of TSP1 expression in human leiomyoma surgical specimens and in immortalized leiomyoma and patient-matched myometrial cultures. Laboratory study. Surgical specimens and immortalized leiomyoma and patient-matched myometrial cell lines were analyzed using qRT-PCR and western blot analysis. In untreated leiomyoma surgical specimens, there was a 30.2% decrease in TSP-1 mRNA expression when compared to patient matched myometrium. Conversely, TSP-1 protein was nearly undetectable in myometrial tissue, while TSP1 protein expression in leiomyoma surgical specimens remained elevated. We demonstrated a similar pattern in immortalized cell lines. In order to determine if we could alter TSP1 expression in leiomyoma cells to patterns comparable to myometrial cells, we treated leiomyoma cells with the retinoic acid metabolic blocking agent liarozole at concentrations ranging from 10−9M to 10−6M. Compared to untreated leiomyoma cells, leiomyoma cells treated with liarozole demonstrated an additional 35% decrease in TSP1 mRNA expression. Leiomyoma surgical specimens had decreased TSP1 gene expression but much greater TSP1 protein expression compared to patient matched myometrium, a pattern mirrored by in vitro cultures. Treatment with liarozole decreased mRNA expression further. Inhibition of TSP1 expression could disrupt both the fibrotic process and angiogenesis, resulting in potential improvement of leiomyoma bulk symptoms.

Four-agents decoction inhibits leiomyoma cell growth

To investigate the efficacy of Four-Agents-Decoction treatment, a Chinese herb commonly used to relieve dysmenorrhea in Taiwan, on human uterine leiomyoma growth. In vitro and animal model experimental study. A nude mice leiomyoma model was used to test the effect of Four-Agents-Decoction on leiomyoma grow. Human leiomyoma cells and myometrial cells were cultured and grown from a pathology proved myoma or normal myometrium respectively. These myometrium cell cultures and leiomyoma cell cultures were proved by histopathology study. The myomectrium and leiomyoma cells were treated with Four-Agents-Decoction. Cell growth was measured by MTT test. Immunohistochemistry study with anti-smooth mucle actin to measure the actin amount in the cells. In animal model study, leiomyoma cube was implanted subcutaneously into the back of nude mice. Tumor size was measured after treatment. Histopathology study revealed that there are less smooth muscle actin in the leiomyoma cells than in the myometrial cells. Serum response factor (SRF), a transcriptional factor crucial for differentiation of muscle progenitor cells to function and normally expressed in myometrial cells, was hardly detected in leiomyoma .Myometrial and leiomyoma cells won't grow in the absence of estrogen. Four-Agents-Decoction suppressed the leiomyoma cell growth. Statistically significant growth inhibition of leiomyoma cells occurred at 10 mg/mL. Immunohistochemistry study revealed all-transretinoic acid treatment increased smooth muscle actin in leiomyoma cells. In nude mice model, Four-Agents-Decoction decreased 30% to 50% of leiomyoma tumor size. Four-Agents-Decoction suppresses leiomyoma cell growth in vitro and in animal model. The nude mice leiomyoma model is a feasible one for futher studying the drug effects on leiomyoma growth.

The role of mast cells and angiogenesis in benign and malignant neoplasms of the uterus

In this study, we looked for a relationship between the extent of angiogenesis and mast cell density (MCD) in human leiomyomas and endometrial carcinomas (EC), and investigated the clinicopathological relevance of mast cells (MCs) in EC. Specimens of 15 control, 20 leiomyoma, and 23 EC patients were investigated immunohistochemically using anti-CD31 and anti-tryptase antibodies. In EC, both stromal and myometrial expressions of CD31 were significantly higher than in the controls ( p < 0.01 and p = 0.013; respectively). Stromal tryptase expression was not significantly lower than that of leiomyoma. In addition, in the leiomyoma group, CD31 and tryptase expressions were not much different compared to the controls. Moreover, a correlation was detected between cancer histological grade and both stromal and myometrial expressions of CD31 ( p = 0.017 and p = 0.005; respectively). The findings show that high grade EC has a higher degree of vascularization than EC of lower grade, but MCD does not increase in parallel with the histological grade. This study has demonstrated that MCD does not correlate with angiogenesis and progression of grade in EC. Moreover, MCD in EC was found to be lower than in benign lesions of the uterus. In conclusion, MCs may not account for the angiogenic process which facilitates tumor growth.

243. Uterine Fibroids Gene Therapy: Targeted Adenovirus Vector (Ad-SSTR-RGD-TK/GCV) Provides Superior Inhibition of Human Leiomyoma Cells than Human Uterine Smooth Muscle Cells

243. Uterine Fibroids Gene Therapy: Targeted Adenovirus Vector (Ad-SSTR-RGD-TK/GCV) Provides Superior Inhibition of Human Leiomyoma Cells than Human Uterine Smooth Muscle Cells

1,25-Dihydroxyvitamin D3 Reduces TGF-β3-Induced Fibrosis-Related Gene Expression in Human Uterine Leiomyoma Cells

Uterine leiomyomas (fibroids) are the most common benign estrogen-dependent tumors of premenopausal women. TGF-β3 up-regulates the synthesis of many of extracellular matrix proteins that are associated with tissue fibrosis. To examine the effect of 1,25-dihydroxyvitamin D(3) (vitamin D(3)) on TGF-β3-induced fibrosis-related protein expression in immortalized human uterine leiomyoma (HuLM) cells. HuLM cells were treated with TGF-β3 with or without vitamin D(3). Western blot analyses were employed to test the effect of vitamin D(3) on TGF-β3-induced protein expression of collagen type 1, fibronectin, and plasminogen activator inhibitor-1 proteins. Western blots as well as immunofluorescence analyses were used to verify the effect of vitamin D(3) on TGF-β3-induced Smad activation involved in extracellular matrix protein synthesis and deposition, which ultimately lead to tissue fibrosis. We observed that TGF-β3 induced fibronectin and collagen type 1 protein expression in HuLM cells, and that effect was suppressed by vitamin D(3). TGF-β3 also induced protein expression of plasminogen activator inhibitor-1, an important TGF-β target, in HuLM cells, which was also inhibited by vitamin D(3). Additionally, TGF-β3 induced phosphorylation of Smad2 as well as nuclear translocation of Smad2 and Smad3 in HuLM cells, whereas vitamin D significantly reduced all these TGF-β3-mediated effects. Therefore, our results suggest that vitamin D(3) has consistently reduced TGF-β3 effects that are involved in the process of fibrosis in human leiomyoma cells. Vitamin D(3) is an antifibrotic factor that might be potentially useful as a novel therapeutic for nonsurgical treatment of benign uterine fibroids.

Involvement of proteasome β1i subunit, LMP2, on development of uterin leiomyosarcma

Background:Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone; however, the development of human uterine leiomyosarcoma is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine leiomyosarcoma from benign tumor leiomyoma is yet to be established.Aims:It is necessary to analyze risk factors associated with human uterine leiomyosarcoma, in order to establish a diagnostic-biomarker and a clinical treatment method.Patients and Methods:Histology and Immunofluorescence Staining: Uteri obtained from LMP2–/– mice or its parental mice (C57BL/6 mice) were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections (5 μm) were prepared and stained with H&E for routine histological examination or were processed further for immunofluorescence staining with appropriate antidodies. Furthermore, a total of 101 patients between 32 and 83 years of age and diagnosed as having smooth muscle tumors of the uterus were selected from pathological files. Immunohistochemistry staining for LMP2 was performed on serial human uterine leiomyosarcoma, leiomyoma and myometrium sections.Results:Homozygous deficient mice for a proteasome β1i subunit, LMP2 spontaneously develop uterine leiomyosarcoma, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine leiomyosarcoma was demonstrated, but present in human leiomyoma and myometrium.Conclusions:Loss in LMP2 expression may be one of the risk factors for human uterine leiomyosarcoma. LMP2 may be a potential diagnostic-biomarker and targeted-molecule for a new therapeutic approach.

Molecular Approach to Uterine Leiomyosarcoma: LMP2-Deficient Mice as an Animal Model of Spontaneous Uterine Leiomyosarcoma

Uterine leiomyosarcoma (LMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker which distinguishes malignant LMS from benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS, in order to establish a treatment method. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. We found LMP2 expression to be absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is a potential diagnostic-biomarker for uterine LMS, and may be targeted-molecule for a new therapeutic approach.

Immunohistochemical analysis of collagen expression in uterine leiomyomata during the menstrual cycle

To investigate the possible involvement of collagen in the characteristic features of human leiomyoma, type I, III and IV collagen expression was determined at the protein level in normal myometrium and leiomyoma tissues throughout the menstrual cycle. The tissues were obtained from 40 pre-menopausal women (29-53 years of age) at various stages of the menstrual cycle who were undergoing abdominal hysterectomy for symptomatic uterine leiomyoma. Immunohistochemical staining was performed with specific monoclonal antibodies against type I, III and IV collagen in the leiomyoma and the myometrial tissues. Immunohistochemical analysis revealed that type I collagen expression was increased in the leiomyoma tissues at the protein level as compared to that in the normal myometrium tissues throughout the menstrual cycle. These results suggest that increased expression of type I collagen plays a key role in the pathogenesis of uterine leiomyoma.