Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.     

Dual repressive effect of angiotensin II-type 1 receptor blocker telmisartan on angiotensin II-induced and estradiol-induced uterine leiomyoma cell proliferation

BACKGROUND: Although uterine leiomyomas or fibroids are the most common gynecological benign tumor and greatly affect reproductive health and well-being, the pathophysiology and epidemiology of uterine leiomyomas are poorly understood. Elevated blood pressure has an independent, positive association with risk for clinically detected uterine leiomyoma. Angiotensin II (Ang II) is a key biological peptide in the renin-angiotensin system that regulates blood pressure. METHODS: In this study, we investigated the potential role of Ang II (1-1000 nM) in the proliferation of rat ELT-3 leiomyoma cells in vitro. RT-PCR and western blot analysis with cell proliferation and DNA transfection assays were performed to determine the mechanism of action of Ang II. RESULTS: Ang II induced ELT-3 leiomyoma cell proliferation (P < 0.01) and the expression of Ang II type 1 receptor (AT(1)R) and AT(2)R mRNA and protein was confirmed. Regarding the intracellular signaling pathway, the Ang II-induced cell proliferation was AT(1)R-, epidermal growth factor receptor-, extracellular-regulated kinase- and protein kinase C-dependent but was not dependent on the AT(2)R or phosphatidylinositol-3 kinase or JAK kinase. The AT(1)R blocker telmisartan, effectively repressed Ang II-induced and estradiol-induced cell proliferation (P < 0.01). AT(1)R, but not AT(2)R, plays a role in Ang II-induced ELT-3 cell proliferation. CONCLUSIONS: These experimental findings in vitro highlight the potential role of Ang II in the proliferation of leiomyoma cells.     

The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells

Uterine fibroids (leiomyomas) are a major women's health problem. Currently, the standard for treatment remains hysterectomy, since no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are benign neoplasias of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by CCN5, a growth arrest-specific gene in vascular smooth muscle cells which is induced and maintained by heparin treatment. Using autologous human myometrial and leiomyoma smooth muscle cells, we demonstrate that the proliferation and motility of both cell types are inhibited by the overexpression of CCN5. Surprisingly, we show that even though CCN5 is induced by heparin in vascular smooth muscle cells, treatment with heparin does not induce CCN5 expression in human uterine smooth muscle cells. Furthermore, we examine CCN5 mRNA expression in 10 autologous pairs of human myometrial and leiomyoma tissues and determine that CCN5 is down-regulated in 100% of the leiomyoma tissues analysed when compared to their normal myometrial counterparts. Thus, our data strongly suggest that CCN5 may exert an important function in maintaining the normal uterine phenotype and that loss of the anti-proliferative protein CCN5 from normal myometrium may account, at least in part, for tumorigenesis.     

A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. METHODS: The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. CONCLUSION: Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.     

A role of growth factors in development of various histological types of uterine leiomyoma

The following growth factors play a role in the development of uterine leiomyoma: EFR- epidermal growth factor, EFR-R- receptor of EFR, TGF- thrombocytic growth factor, TGFb- transforming growth factor beta, ILGF1 - insulin-like growth factor of the 1st type. These factors stimulate proliferation of cells, stroma and angiogenesis in the tumor. Mitotically active leiomyoma is characterized by a higher content of these growth factors vs ordinary and cellular leiomyomas. Treatment of uterine leiomyoma should be concentrated on removal of tumor cells from the body as leiomyomas especially its mitotically active variant produce growth factors which are able to stimulate tumor growth by the autocrine mechanism.     

Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-alpha and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle-specific cytoskeletal protein alpha-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.     

Activation of peroxisome proliferator-activated receptor-gamma by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells

During hepatic fibrogenesis, reduction in the abundance of peroxisome proliferator-activated receptor-gamma (PPARgamma) is accompanied by activation of mitogenic signaling for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in hepatic stellate cells (HSCs), the major effector cells. We previously reported that curcumin, the yellow pigment in curry, interrupted PDGF and EGF signaling, stimulated PPARgamma gene expression, and enhanced its activity, leading to inhibition of cell proliferation of activated HSC in vitro and in vivo. The aim of this study was to elucidate the underlying mechanisms. We hypothesized that the enhancement of PPARgamma activity by curcumin might result in the interruption of PDGF and EGF signaling. Our experiments demonstrated that curcumin, with different treatment strategies, showed different efficiencies in the inhibition of PDGF- or EGF-stimulated HSC proliferation. Further experiments observed that curcumin dose dependently reduced gene expression of PDGF and EGF receptors (ie, PDGF-betaR and EGFR), which required PPARgamma activation. The activation of PPARgamma by its agonist suppressed pdgf-betar and egfr expression in HSC. In addition, curcumin reduced the phosphorylation levels of PDGF-betaR and EGFR, as well as their downstream signaling cascades, including ERK1/2 and JNK1/2. Moreover, activation of PPARgamma induced gene expression of glutamate-cysteine ligase, the rate-limiting enzyme in de novo synthesis of the major intracellular antioxidant, glutathione. De novo synthesis of glutathione was required for curcumin to suppress pdgf-betar and egfr expression in activated HSCs. Our results collectively demonstrated that enhancement of PPARgamma activity by curcumin interrupted PDGF and EGF signaling in activated HSCs by reducing the phosphorylation levels of PDGF-betaR and EGFR, and by suppressing the receptor gene expression. These results provide novel insights into the mechanisms of curcumin in the inhibition of HSC activation and the suppression of hepatic fibrogenesis.     

Cell proliferation and apoptosis in human uterine leiomyomas and myometria

To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis -- Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) -- and two endogenous markers of cell replication - proliferating cell nuclear antigen (PCNA) and Ki-67 - in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly ( P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.     

The influence of extracellular matrix proteins on T-cell proliferation and apoptosis in women with endometriosis or uterine leiomyoma

PROBLEM: Interactions between the extracellular matrix (ECM) and peripheral blood T cells in women with endometriosis and leiomyoma are hardly unknown. We have investigated the influence of two major ECM components, collagen IV (C-IV) and fibronectin (Fn), on T-cell proliferation and apoptosis in women with endometriosis and uterine leiomyoma. beta1 integrin expression, responsible for interactions with ECM proteins, was also studied. METHOD OF STUDY: Peripheral blood lymphocytes were obtained from 53 women (17 with uterine leiomyomas, 18 with endometriosis, and 18 from healthy donors). T cells were exposed to ECM proteins co-immobilized with monoclonal antibody anti-CD3 for 72 hr. Apoptosis and S phase of the cell cycle of the T cells were studied by DNA analysis using flow cytometry. The proliferation of T cells was evaluated by MTT assay. The percentage of CD3+ cells expressing CD29 (beta1 integrin chain) was evaluated by double-color flow cytometry. Results were analyzed statistically using the Mann-Whitney test. RESULTS AND CONCLUSIONS: (1) A general increase in the percentage of T cells in S phase could be seen in women with endometriosis and uterine leiomyoma in all culture conditions what may suggest general activation of T cells. (2) A significant increase in the percentage of cells in S phase was shown only in the case of T cells exposed to anti-CD3 + C-IV in both women with uterine leiomyoma and endometriosis. (3) However, no apoptotic cells were observed. (4) T cells from patients with uterine leiomyoma exhibited significantly increased level of proliferation after culture with anti-CD3 + C-IV. (5) More T cells expressed beta1 integrin in women with endometriosis or uterine leiomyoma than in healthy donors. Our data may suggest that increased beta1 integrin expression may enhance T-cell-ECM interactions, which may be responsible for the increased proliferation of T cells but not for apoptosis. Therefore, it is possible that interactions of T cells with ECM proteins, especially with C-IV, may contribute to the pathogenesis of endometriosis and uterine leiomyoma.     

Relationship between platelet-derived growth factor expression in leiomyomas and uterine volume changes after gonadotropin-releasing hormone agonist treatment

The unopposed estrogen effect is the main cause of leiomyoma growth and is at the basis of the clinical use of gonadotropin-releasing hormone (GnRH) agonists. Platelet-derived growth factor (PDGF) has been indicated as the main growth factor involved, in vitro in the proliferation response of leiomyoma smooth muscle cells to estrogen stimulation. The aim of this article is to evaluate the mitogenic action of PDGF in vivo by studying the relationship between PDGF expression in leiomyomas and post-GnRH analogue treatment changes in uterine volume. Thirty-nine patients suffering from uterine leiomyomas were treated with leuprorelin acetate depot 3.75 mg for three cycles; 31 untreated patients were enrolled as control group. Uterine volume was determined twice by ultrasonography in each patient, the first time at admission and the second time after treatment in the study group and after 3 months in the control group. The change in the uterine volume was then evaluated. Patients underwent surgery, and PDGF immunohistochemical detection was performed on the obtained fibroid samples. Uterine volume decreased significantly after treatment, whereas just a poor modification was found in the controls. The decrease in the uterine volume was found to be statistically related to PDGF expression. Thus PDGF levels decreased in treated patients as compared with controls. The decreased PDGF production in leiomyomas after GnRH analogue treatment and the relationship between decreased PDGF expression and greater shrink age in uterine volume suggest that PDGF might have a mitogenic action on leiomyomas in vivo.     

Cyclin-dependent kinase inhibitor p27Kip1 controls growth and cell cycle progression in human uterine leiomyoma

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27(Kip1) (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometrium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma.     

Peroxisome proliferator-activated receptor-gamma ligands inhibit alpha5 integrin gene transcription in non-small cell lung carcinoma cells

We previously showed that fibronectin stimulates the growth of non-small cell lung carcinoma (NSCLC) cells through integrin alpha5beta1-dependent signals. We also demonstrated that peroxisome proliferator-activated receptor (PPAR)gamma ligands inhibit lung carcinoma cell growth. Because alpha5beta1 activation elicits cellular signals linked to cell survival and regulation of cell cycle progression, we studied the effects of PPARgamma ligands on its expression. We found that PPARgamma ligands decreased mRNA and protein expression of the alpha5 subunit of the alpha5beta1 heterodimer in NSCLC; this was associated with reduced NSCLC adhesion to fibronectin. The suppressive effect of the PPARgamma ligands BRL 49653 and GW1929, but not PGJ(2), on alpha5 gene expression were reversed by GW9662, an antagonist of PPARgamma. GW1929 activated the extracellular regulated kinase (Erk), and an inhibitor of the Erk pathway (PD98095) prevented its effect on alpha5. PPARgamma ligands also reduced alpha5 gene promoter activity, and this was blocked by Erk antisense oligonucleotides. PPARgamma ligands GW1929 and BRL49653 inhibited AP-1 DNA binding, whereas 15d-PGJ(2) inhibited Sp1 DNA binding; both effects were blocked by Erk antisense oligonucleotides. GW1929 partially blocked fibronectin-induced NSCLC cell growth, but did not affect cell growth induced by epidermal growth factor. These results suggest that PPARgamma ligands inhibit alpha5 expression in NSCLC through Erk-related signals.     

The AG1478 tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth

BACKGROUND: Uterine leiomyomas are the most common benign smoothmuscle cell tumours in women. Formation of leiomyomas, stillnot completely understood, is viewed as a multistep process,with involvement of ovarian steroid hormones, cytokines andgrowth factors. Our study aimed to identify tyrosine kinaseinhibitors as potential ‘signal transduction therapeutics’for leiomyomas, underlying the effect of ovarian steroidal hormones.METHODS: The selective epidermal growth factor (EGF) receptorblocker AG1478 was evaluated as a potential target, since EGFhas been shown to mediate estrogen action and to play a crucialrole in regulating leiomyoma growth. Paired cultures of leiomyomaand normal myometrium samples were established and the suppressiveeffect of AG1478 on the cells prior and subsequent to steroidalhormone treatment was examined: cell proliferation, recoveryafter treatment, cell cycle analysis and immunochemical analysisof relevant proteins. RESULTS: Leiomyoma cell growth is effectivelyblocked by AG1478 and is unaffected by the presence of physiologicalconcentrations of progesterone and estradiol. AG1478 (10 µM)completely suppressed proliferation and the cells did not recoverafter cessation of treatment. CONCLUSION: The growth-arrestingproperties of AG1478, unaffected by ovarian steroidal hormones,identify it as a potential lead agent for the non-surgical managementof uterine leiomyomas.     

Uterus and endometrium: In-vitro model of uterine leiomyomas: formation of ball-like aggregates

To clarify the biological characteristics of uterine leiomyomas,cells explanted and cultured from uterine leiomyomas and fromnormal myometrlal tissue were observed by time- lapse cinemicrographyand phase-contrast microscopy. The histological characteristicswere evaluated by electron microscopy and immunofluorescencemicroscopy, and these observations revealed significant differences.By time-lapse cinemicrography, the cells cultured from leiomyomasand myometriuin differed in their behaviour. Cells from themyometrium started to grow in parallel with the cell's majoraxis and formed topographically uniform hills and valleys byday 21 of culture. In contrast, the cells from leiomyomas startedto grow irregularly, as if having no contact inhibition, andformed ball-like aggregates of cells by day 21 of culture. Theaggregates resembled the nodules of leiomyoma in vivo. Ultrastructurally,cells from both leiomyomas and myometrium had typical featuresof smooth muscle. Immunofluorescently, a different distribution of -smooth muscle actin-positive filaments and differentstaining of cellular fibronectin and N-cadherin between thecells from Ieiomyomas and myometrium were observed, which maycontribute in part to the different behaviour of the cells.Given that the explant cell culture system resembles the featuresof uterine leiomyomas in vivo, this suggests that it can beused as an in-vitro model.     

A uterine leiomyoma showing both t(12;14) and del(7) abnormalities

The involvement of chromosomes 12 and 14 in uterine leiomyomas has been well established. However, in a recent report of only a del(7)(q22.1q31.32) or (q11.2q22.3) in two cases of typical uterine leiomyoma, Boghosian et al. hypothesized that this could represent a cytogenetic subgroup of uterine leiomyomas. We report a case of uterine leiomyoma with both the t(12;14) and del(7) in all the cells examined and discuss the implications of this in terms of critical chromosomal rearrangements underlying the route to benign cellular proliferation.     

Potential link between estrogen receptor-alpha gene hypomethylation and uterine fibroid formation

Uterine leiomyomas are the most common uterine tumors in women. Estrogen receptor-alpha (ER-alpha) is more highly expressed in uterine leiomyomas than in normal myometrium, suggesting a link between uterine leiomyomas and ER-alpha expression. DNA methylation is an epigenetic mechanism of gene regulation and plays important roles in normal embryonic development and in disease progression including cancers. Here, we investigated the DNA methylation status of the ER-alpha promoter region (-1188 to +229 bp) in myometrium and leiomyoma. By sodium bisulfite sequencing, 49 CpG sites in the proximal promoter region of ER-alpha gene were shown to be unmethylated in both leiomyoma and normal myometrium. At seven CpG sites in the distal promoter region of the ER-alpha gene, there was a variation in DNA methylation status in myometrium and leiomyoma. Further analysis of the DNA methylation status by bisulfite restriction mapping among 11 paired samples of myometrium and leiomyoma indicated that CpG sites in the distal region of ER-alpha promoter are hypomethylated in leiomyomas of nine patients. In those patients, ER-alpha mRNA levels tended to be higher in the leiomyoma than in the myometrium. In conclusion, there was an aberrant DNA methylation status in the promoter region of ER-alpha gene in uterine leiomyoma, which may be associated with high ER-alpha mRNA expression.