interleukin-8 and Leiomyoma

To determine the mechanism by which tranilast induces miR-200c expression in leiomyoma smooth muscle cells (LSMCs).. Experimental study.. Academic research laboratory.. Women undergoing hysterectomy for leiomyoma.. Blockade of RelA/p65.. Effects of tranilast and blockade of RelA/p65 on miR-200c expression.. Tranilast, an inflammation inhibitor, dose-dependently induced miR-200c in LSMCs and myometrium smooth muscle cells (MSMCs), with a more profound effect in LSMCs than in MSMCs. The treatment of LSMCs with Bay 117082, an inhibitor of IκB phosphorylation, further enhanced miR-200c induction by tranilast. The knockdown of RelA/p65 by small interfering RNA also induced miR-200c expression in LSMCs. Although tranilast had no effect on total RelA/p65 protein levels in LSMCs, it significantly induced RelA/p65 phosphorylation at S536 while reducing its activity as well as its nuclear translocation. ChIP assay indicated that tranilast reduces the binding ability of RelA/p65 to miR-200c promoter, resulting in miR-200c induction. Tranilast also inhibited interleukin-8 (IL8) expression in LSMCs. The induction of miR-200c by tranilast partially mediates the inhibitory effect of tranilast on the expression of IL8 and cyclin-dependent kinase 2 in LSMCs.. Induction of miR-200c by tranilast in LSMCs is mediated through a transcriptional mechanism involving inhibition of the nuclear factor κB signaling pathway. These results highlight the significance of inflammation in the pathogenesis of leiomyoma and the potential utility of antiinflammatory drugs for treatment of leiomyomas.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cyclin-Dependent Kinase 2; Female; Humans; Interleukin-8; Leiomyoma; MicroRNAs; Myocytes, Smooth Muscle; Myometrium; ortho-Aminobenzoates; Signal Transduction; Transcription Factor RelA; Tumor Cells, Cultured; Uterine Neoplasms

We analyzed cytokine profile in blood serum of patients with uterine myoma and revealed significantly reduced level of IFNγ and a tendency towards a decrease in the levels of IL-1β and TNFα; the levels of IL-6, IL-8, and IL-12p70 did not differ from those in healthy women. The drop in the concentrations of factors responsible for inflammation and angiogenesis in tissues are unfavorable for proliferation and differentiation of the uterine tissues.

Topics: Adult; Female; Gene Expression; Humans; Inflammation; Interferon-gamma; Interleukin-12; Interleukin-1beta; Interleukin-6; Interleukin-8; Leiomyoma; Middle Aged; Tumor Necrosis Factor-alpha; Uterine Myomectomy; Uterine Neoplasms; Uterus

We have previously reported that leiomyoma expressed lower levels of miR-200c and elevated IL8 as compared to paired myometrium. Here we addressed the regulatory functions of miR-200c on the expression of inflammatory mediators and cellular viability using leiomyomas and paired myometrium and their isolated primary smooth muscle cells. Our results indicated that gain-of function or knockdown of miR-200c in leiomyoma smooth muscle cells (LSMC) regulated IL8 mRNA and protein expression through direct targeting of IKBKB and alteration of NF-kB activity. Additionally, leiomyoma expressed higher levels of phosphorylated IKBKB with no significant difference in the level of IKBKB mRNA and protein as compared to matched myometrium. Gain-of function of miR-200c in LSMC resulted in decreased IkBα phosphorylation and p65 nuclear translocation, which led to decreased p65 transcriptional activity of IL8 promoter, and increased caspase 3/7 activity which was not reversible following IL8 restoration. Collectively, our results suggest that NF-κB signaling pathway is a target of miR-200c regulatory function, and low level of miR-200c expression in leiomyoma by transcriptional regulation of inflammatory mediators such as IL8, in part account for development of leiomyomas.

Topics: Adult; Base Sequence; Caspase 3; Caspase 7; Down-Regulation; Gene Expression Regulation; Humans; I-kappa B Kinase; Inflammation; Interleukin-8; Leiomyoma; MicroRNAs; Middle Aged; Molecular Sequence Data; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Transcription Factor RelA

We investigated the potential of gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists to inhibit cell proliferation in endometriotic and endometrial stromal cells.. Twenty patients with ovarian endometriomas and 18 patients with uterine fibromas were recruited. Endometriotic and endometrial stromal cells were obtained from the ovarian chocolate cyst linings and the eutopic endometria of premenopausal women with uterine fibromas, respectively.. GnRH agonist or antagonist treatment attenuated tumor necrosis factor (TNF)-α-induced cell proliferation in the endometrial stromal cells, whereas endometriotic stromal cells did not respond to treatment. The endometriotic stromal cells exhibited a decreased expression of the type I GnRH receptor compared with the endometrial stromal cells. GnRH agonists or antagonists did not repress TNF-α-induced IL-8 production in endometriotic stromal cells.. GnRH agonists and antagonists have similar effects in slowing the growth of endometrial stromal cells. Endometriotic stromal cells resist the antiproliferative effect of GnRH agonists and antagonists.

Topics: Adult; Blotting, Western; Buserelin; Cell Proliferation; Endometriosis; Endometrium; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Gonadotropin-Releasing Hormone; Humans; Interleukin-8; Leiomyoma; Ovarian Diseases; Premenopause; Receptors, LHRH; Stromal Cells; Tumor Necrosis Factor-alpha; Uterine Neoplasms

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Topics: Adult; Caspases; Cell Cycle Proteins; Cell Proliferation; Cell Survival; Cells, Cultured; Cluster Analysis; DNA-Binding Proteins; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Leiomyoma; Menstrual Cycle; MicroRNAs; Middle Aged; Minichromosome Maintenance Complex Component 7; Myometrium; Nuclear Proteins; RNA Interference; Thromboplastin; Uterine Neoplasms; Young Adult

We previously reported that lipopolysaccharide (LPS)-promoted endometriotic stromal cell (ESC) proliferation by inducing TNFalpha production. The aim of this study was to investigate the efficacy of TNFalpha gene silencing on LPS-treated ESCs.. Endometriotic stromal cells (ESCs) and endometrial stromal cells (ESCs) (EMSCs) were obtained from ovarian chocolate cysts and uterine myoma, respectively. Using PCR array, LPS-induced gene expression profiling after transfection of TNFalpha siRNA into ESCs was performed. Down-regulated genes by TNFalpha silencing were examined using real-time RT-PCR. Effect of TNFalpha silencing was examined using ELISA and BrdU incorporation, respectively.. In PCR array, TNFalpha silencing in ESCs repressed LPS-induced expression of cIAP2 and IL-8, NFkappaB pathway responsive genes. After adding LPS, the levels of cIAP2 and IL-8 expression in ESCs were higher compared with those in EMSCs. TNFalpha silencing attenuated the LPS-induced ESC proliferation.. Tumor necrosis factor alpha may be involved in cell proliferation of endometriotic tissues.

Topics: Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; Cell Proliferation; Cells, Cultured; Endometriosis; Endometrium; Female; Gene Expression Profiling; Gene Silencing; Humans; Inhibitor of Apoptosis Proteins; Interleukin-8; Leiomyoma; Lipopolysaccharides; NF-kappa B; Ovarian Cysts; Ovarian Diseases; RNA, Small Interfering; Signal Transduction; Stromal Cells; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases; Uterine Neoplasms

To investigate whether certain polymorphisms are correlated with leiomyoma susceptibility, i.e., interleukin (IL)-1, IL-2, IL-4, IL-8, IL-12, and IL-18, which are all immunomodulatory cytokines that play important roles in host immune responses against cancers.. Departments of gynecology and genetics in a medical center.. Women were divided into: [1] a leiomyoma group (n = 162) and [2] a nonleiomyoma group (n = 156).. Genotyping for the IL-1beta-511 promoter, IL-1beta exon 5, IL-1Ra, IL-2 114, IL-4 -590 intron 3, IL-8 3'-UTR 2767, IL-12Rbeta1 codon 378, and IL-18 105 were evaluated by polymerase chain reaction-restriction fragment length polymorphism.. Genotypes and allelic frequencies in both groups were compared.. Proportions of IL-12Rbeta1 codon 378 *CC/CG/GG in the leiomyoma and nonleiomyoma groups were: [1] 7.4%/43.8%/48.8% and [2] 11.5%/54.5%/34%, respectively. Distributions of other polymorphisms in both groups were not significantly different. Proportions of IL-1beta-511 promoter *CC/CT/TT were: [1] 22.8%/50%/27.2% and [2] 21.8%/57.1%/21.1% in the leiomyoma and nonleiomyoma groups, respectively. The IL-1beta exon 5 *E1 homozygote/heterozygote/E2 homozygote were: [1] 96.3%/3.7%/0% and [2] 96.9%/3.1%/0% in the leiomyoma and nonleiomyoma groups, respectively. Alleles I/II/III/IV/V for IL-1Ra were: [1] 92.6%/7.1%/0.3%/0/0% and [2] 93.9%/5.7%/0%/0.4/0% in the leiomyoma and nonleiomyoma groups, respectively. The IL-2 114 G homozygote/heterozygote/T homozygote were: [1] 27.8%/49.4%/22.8% and [2] 20.5%/53.2%/26.3% in the leiomyoma and nonleiomyoma groups, respectively. The IL-4 -590 intron 3 *RP1 homozygote/heterozygote/RP2 homozygote were: [1] 64.8%/32.7%/2.5% and [2] 69.2%/26.9%/3.9% in the leiomyoma and nonleiomyoma groups, respectively. The IL-8 3'-UTR 2767 A homozygote/heterozygote/G homozygote were: [1] 14.2%/43.8%/42% and [2] 20.5%/41.7%/37.8% in the leiomyoma and nonleiomyoma groups, respectively. The IL-18 *AA/AC/CC were: [1] 56.8%/40.7%/2.5% and [2] 59%/39.7%/1.3% in the leiomyoma and nonleiomyoma groups, respectively.. The IL-12Rbeta1 codon 378 *G homozygote and G allele are related to a higher susceptibility to leiomyoma. The IL-1beta-511 promoter, IL-1beta exon 5, and IL-1Ra, IL-2 114, IL-4 -590 intron 3, IL-8 3'-UTR 2767, and IL-18 105 gene polymorphisms are not correlated with the development of leiomyoma.

Topics: 3' Untranslated Regions; Codon; Exons; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-18; Interleukin-1beta; Interleukin-2; Interleukin-4; Interleukin-8; Leiomyoma; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Receptors, Interleukin-12; Uterine Neoplasms

To evaluate the involvement of chemokines in the pathogenesis of endometriosis, we investigated the expression of CXC chemokines in cultured ovarian endometriotic cyst stromal cells (ECSC), endometrial stromal cells with endometriosis (ESCwE), and normal endometrial stromal cells (NESC). Using ELISA, TNF-alpha significantly enhanced the production of IL-8, growth-related oncogene alpha, and epithelial neutrophil-activating peptide-78 in all cases of ECSC (n = 10), ESCwE (n = 6), and, NESC (n = 10). IL-1beta did not affect the production of these chemokines in eight of 10 cases of ECSC. In contrast, IL-1beta significantly enhanced the expression of these chemokines in all cases of ESCwE (n = 6) and NESC (n = 10). Western blot analysis revealed down-regulation of expression of IL-1 receptor type 1 (IL-1-R1) in all cases of ECSC with low response to IL-1beta (n = 8). In contrast, significant IL-1-R1 expression was detected in all cases of NESC. Although IL-1-R1 expression was detected in all cases of ESCwE (n = 6), its expression in ESCwE tended to decrease compared with that in NESC. Moreover, phosphorylation of inhibitor kappaB-alpha was detected in all cases of ESCwE and NESC after stimulation with IL-1beta, but not in ECSC with low response to IL-1beta (n = 8). In contrast, significant IL-1-R2 expression was detected in all cases of ECSC, ESCwE, and NESC. The present findings suggest that the dysregulation of IL-1/IL-1-R system relates to immunological dysfunction in endometriosis. The alteration of the CXC chemokines expression may be important for elucidation of the pathogenesis of endometriosis.

Topics: Chemokine CXCL1; Chemokine CXCL5; Chemokines, CXC; Down-Regulation; Endometrial Stromal Tumors; Endometriosis; Female; Gene Expression; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Leiomyoma; Receptors, Interleukin-1; Receptors, Interleukin-1 Type I; Receptors, Interleukin-1 Type II; Receptors, Tumor Necrosis Factor, Type I; RNA, Messenger; Signal Transduction; Stromal Cells; Tumor Cells, Cultured

Interleukin 8 is a potent chemoattractant cytokine that is expressed in a variety of human tumors and is known to induce mitogenesis. We aimed to investigate the production of interleukin 8 and the expression of its receptor in myometrium and leiomyoma, in which we hypothesized that interleukin 8 may contribute to cellular proliferation.. Myometrial and leiomyoma tissue pairs (n = 14) were obtained from human uteri after hysterectomy conducted for leiomyomatous uterus. Expression of interleukin 8 and interleukin 8 receptor type A was identified in the leiomyomatous myometrium by means of specific antibodies directed against interleukin 8 and interleukin 8 receptor type A for immunohistochemical detection. Interleukin 8 production by cultured cells was measured by enzyme-linked immunosorbent assay. The regulation of interleukin 8 messenger ribonucleic acid expression was assessed by means of the Northern blot analysis after treatment of myometrial cells with interleukin 1alpha and tumor necrosis factor alpha. Myometrial cell proliferation was determined by means of colorimetric assay after cells were treated with interleukin 8 and antihuman interleukin 8 neutralizing antibody.. Immunostaining for both interleukin 8 and interleukin 8 receptor type A was stronger in the myometrium adjacent to leiomyoma compared with leiomyoma itself (2-fold, P <.05). Compared with samples from nonusers, samples from patients who had used gonadotropin-releasing hormone agonists revealed a trend for decreased staining for both interleukin 8 and interleukin 8 receptor type A. Interleukin 1alpha and tumor necrosis factor alpha caused a time- and dose-dependent increase in interleukin 8 production by myometrial cells (P <.001). There was a dose-dependent inhibition of cell proliferation with antihuman interleukin 8 antibody to 55% of the control (P <.001).. Our demonstration of high levels of interleukin 8 and its receptor in myometrium immediately surrounding leiomyoma and the inhibition of cell proliferation when interleukin 8 is blocked by a neutralizing antibody suggest a potential role for interleukin 8 in the growth of myometrial tissue surrounding leiomyomatous tissue. This study could lead to a better understanding of potential involvement of cytokines in leiomyoma growth and in gonadatropin-releasing hormone agonist-induced regression.

Topics: Blotting, Northern; Cell Division; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Gonadotropin-Releasing Hormone; Humans; Immunohistochemistry; Interleukin-1; Interleukin-8; Kinetics; Leiomyoma; Myometrium; Receptors, Interleukin-8A; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Neoplasms