A botanical extract from channel flow inhibits cell proliferation, induces apoptosis, and suppresses CCL5 in human endometriotic stromal cells.

Growing evidence suggests that medicinal herbs have direct actions on endometrial cells. By screening multiple herbs using an in vitro model of endometriosis, we found that a commonly used herbal formula exerted considerable antiproliferative effects. Our purpose was to investigate the effects of this antiendometriosis herbal mixture on cell proliferation, apoptosis, and CCL5 expression and secretion in endometriotic stromal cells in vitro. Isolated normal endometrial, eutopic, and ectopic endometriotic stromal cells were cultured under established conditions. Cell proliferation, apoptosis, and CCL5 gene expression protein secretion was evaluated after incubation with different concentrations of an antiendometriosis herbal mixture extract. Cell proliferation was assessed by cell counting, (3)H-thymidine incorporation, and MTS assays. Apoptosis was determined by blotting using anti-cleaved caspase 3 antibodies and by a TUNEL assay. CCL5 gene expression and protein secretion were determined by transient transfection of gene promoter reporters and ELISAs in cell supernatants. Extracts of a traditional herbal mixture dose-dependently decreased cell proliferation in normal, eutopic, and ectopic endometriotic stromal cells. (3)H-Thymidine uptake and MTS confirmed these findings. The herbal extracts induced apoptosis, as evidenced by activation of caspase 3 and the presence of TUNEL-positive cells after treatment. The herbal extracts also suppressed CCL5 gene transcription and protein secretion in endometriotic stromal cells, even when corrected for cell number. Extracts from a medicinal herbal mixture have direct effects on cell proliferation, apoptosis, and CCL5 production in endometriotic stromal cells. Our findings support the further investigation of novel, potentially safe and well-tolerated botanical products as future endometriosis treatments.

Sulindac suppresses nuclear factor-kappaB activation and RANTES gene and protein expression in endometrial stromal cells from women with endometriosis.

CONTEXT: The nuclear factor-kappaB (NF-kappaB) pathway is a critical mediator of RANTES (regulated on activation, normal T cell expressed and secreted) gene regulation and therefore represents a potential target for therapy of endometriosis-associated symptoms. OBJECTIVE: The objective of this study was to investigate the effects of the antiinflammatory drug sulindac on NF-kappaB activation, NF-kappaB-mediated gene expression, RANTES gene and protein expression in endometrial stromal cells isolated from women with endometriosis, and unaffected controls. DESIGN: This was a clinical experimental study. SETTING: The study was conducted at a university hospital. RESULTS: The inflammatory response in endometriosis is augmented by a 5-fold increased TNFalpha-induced RANTES secretion from ectopic endometriotic stromal cells, compared with normal endometrial stromal cells (P < 0.05). Western blot analysis revealed basal activation of NF-kappaB in endometriotic cells, which could be suppressed by sulindac. EMSAs showed that sulindac dramatically decreased NF-kappaB activation and diminished TNFalpha and IL-1beta-induced NF-kappaB DNA binding activity. Sulindac pretreatment resulted in a significant decrease in TNFalpha-induced luciferase activity of NF-kappaB response element and -477 bp RANTES promoter constructs in normal and endometriotic stromal cells. The addition of sulindac to IL-1beta- and TNFalpha-treated endometriotic stromal cells also resulted in a 4-fold inhibition of RANTES protein secretion (P < 0.05). CONCLUSIONS: We have demonstrated that sulindac exerts strong antiinflammatory effects by suppression of NF-kappaB translocation, inhibition of NF-kappaB-mediated gene transcription, RANTES gene expression, and protein secretion in normal and endometriotic stromal cells. These results suggest that drugs targeting the NF-kappaB pathway may be beneficial in the treatment of endometriosis-associated symptoms.

Anatomy of the estrogen response element.

Estrogens exert their regulatory potential on gene expression through different nuclear and non-nuclear mechanisms. A direct nuclear approach is the interaction of estrogen with specific target sequences of DNA, estrogen response elements (ERE) or units. EREs can be grouped into perfect and imperfect palindromic sequences with the imperfect sequences differing from the consensus sequence in one or more nucleotides and being less responsive to the activated estrogen-estrogen receptor (ER) complex. Differences in the ERE sequence and the ER subtype involved can substantially alter ER-ERE interaction. In addition, cross-talk between ERs and other nuclear transcription factors profoundly influences gene expression. Here, we focus on the recent advances in the understanding of the structure of EREs and how ERs are recruited to these. Identifying known target genes for estrogen action could help us to understand the potential risks and benefits of the administration of this steroid to humans.

PROGINS receptor gene polymorphism is associated with endometriosis.

OBJECTIVE: To investigate the association between the 306-base pair insertion polymorphism in intron G of the progesterone receptor (PROGINS) and endometriosis. DESIGN: Case-control study. SETTING: Tertiary care center. PATIENT(S): Ninety-five white women with surgically diagnosed and histologically confirmed endometriosis and 107 white women without endometriosis (controls). INTERVENTION(S): Determination of PROGINS was performed by polymerase chain reaction and gel electrophoresis. MAIN OUTCOIME MEASURE(S): Frequency and distribution of the PROGINS allele. RESULT(S): Frequencies of the mutant allele T2 was 0.17 among women with endometriosis and 0.08 among controls (odds ratio, 2.41 [CI, 1.31-4.53]). Homozygosity for allele T2 was present in 3.2% of women with endometriosis and 0.9% of controls. CONCLUSION(S): PROGINS appears to be associated with endometriosis in white persons.

Polymorphism of the interleukin-1beta gene and endometriosis.

OBJECTIVE: Experimental data indicate that interleukin (IL)-1 plays a role in the pathogenesis of endometriosis. We determined the genotype and the allele frequencies of the IL1B exon 5 polymorphism and the corresponding IL-1beta serum levels in women with endometriosis. METHODS: We genotyped 92 women with surgically and histologically confirmed endometriosis and 69 controls without history of endometriosis. Both groups were of middle European genetic background for the IL1B exon 5 polymorphism (at position +3954). Genotyping was performed by pyrosequencing. IL-1beta serum levels were analyzed by a commercially available enzyme-linked immunosorbent assay. RESULTS: Allele frequencies in women with endometriosis and controls were 76.6% and 76.8%, respectively, for the E1 allele (wild type) and 23.4% and 23.2%, respectively, for the E2 allele (polymorphic) (odds ratio 1.01; P > .99). The investigated polymorphism of the IL-1beta gene was not correlated with IL-1beta serum levels. CONCLUSIONS: We did not find an association between the IL1B exon 5 polymorphism, endometriosis, or increased serum IL-1beta levels.

Tumor necrosis factor-alpha promotor polymorphisms and endometriosis.

To explore whether having the mutant tumor necrosis factor (TNF)2 (G-308*A) and TNFA-A (G-238*A) alleles in the TNF-alpha gene promotor region is higher in women with endometriosis, we determined the respective genotype and allele frequencies in a retrospective case-control study. Polymerase chain reaction was performed to identify the G-308A and G-238A promotor polymorphisms in 92 women with surgically and histologically confirmed endometriosis. A series of 69 healthy women without a history of endometriosis served as clinical controls. The allele frequencies of the TNF2 polymorphism were 0.13 and 0.16 in women with endometriosis and in the control group, respectively, and the frequencies of the TNFA-A polymorphisms in women with endometriosis and in the control group were 0.04 and 0.05, respectively, with no significant difference between the study and control groups. The TNF2 polymorphism was present in the homozygous form (TNF(2/2)) in 4.3% of women with endometriosis and in 2.9% of controls (P=.7). No TNFA-A homozygotes (TNFA(A/A)) were detected. We studied TNF-alpha promotor gene variants among women with endometriosis and found that having the G-308A TNF-alpha and the G-238A TNF-alpha polymorphism was not associated with endometriosis in a white population.

Analysis of an interleukin-6 gene promoter polymorphism in women with endometriosis by pyrosequencing.

Interleukin (IL)-6 has been implicated in the etiology of endometriosis. A single nucleotide polymorphism (SNP) at position -174 in the IL-6 gene promoter appears to influence IL-6 transcription rates in vitro and basal IL-6 levels in vivo. We determined the genotype and the allele frequencies of the -174 IL-6 promoter polymorphism and the corresponding IL-6 serum levels in women with endometriosis. The pyrosequencing technique was used to assess the IL-6 genotypes in 94 women with histologically confirmed endometriosis (study group). A series of 70 healthy women without history of uterine disease served as clinical controls (control group).Allele frequencies for the G allele among women with and without endometriosis were 59.6% and 55.0%, respectively (P =.430; odds ratio [OR] 0.83, 95% confidence interval [CI] 0.53, 1.29). Homozygotes for the protective allele C were present in 17.0% of women with endometriosis and in 18.6% of controls were homozygous for the protective allele C (P =.797; OR 0.90, 95% CI 0.40, 2.02). When patients with various disease manifestations were compared, we found an association between the -174 G allele and chocolate cysts (P =.037). Serum levels of IL-6 were significantly higher in women with endometriosis than in controls (P <.001), with highest levels in women with chocolate cysts. There was no association between serum IL-6 levels and IL-6 genotype. The IL-6 promoter polymorphism -174 G/C does not contribute significantly to overall disease susceptibility but does predispose the carrier to distinct endometriosis with chocolate cysts. A genetically determined high IL-6 response might play a pathogenic role in this disease condition.

Advances in the genetics of endometriosis.

Endometriosis is a gynecological disease characterized by implantation of endometrial tissue outside of the uterus. Early familial aggregation and twin studies noted a higher risk of endometriosis among relatives. Studies on the roles of the environment, genetics and aberrant regulation in the endometrium and endometriotic lesions of women with endometriosis suggest that endometriosis arises from the interplay between genetic variants and environmental factors. Elucidating the hereditary component has proven difficult because multiple genes seem to produce a susceptibility to developing endometriosis. Molecular techniques, including linkage and genome-wide analysis, have identified candidate genes located near known loci related to development and regulation of the female reproductive tract. As new candidate genes are discovered and hereditary pathways identified using technologies such as genome-wide analysis, the possibility of prevention and treatment becomes more tangible for millions of women affected by endometriosis. Here, we discuss the advances of genetic research in endometriosis and describe technologies that have contributed to the current understanding of the genetic variability in endometriosis, variability that includes regulatory polymorphisms in key genes.

PPAR Action in Human Placental Development and Pregnancy and Its Complications.

During pregnancy crucial anatomic, physiologic, and metabolic changes challenge the mother and the fetus. The placenta is a remarkable organ that allows the mother and the fetus to adapt to the new metabolic, immunologic, and angiogenic environment imposed by gestation. One of the physiologic systems that appears to have evolved to sustain this metabolic regulation is mediated by peroxisome proliferator-activated receptors (PPARs). In clinical pregnancy-specific disorders, including preeclampsia, gestational diabetes, and intrauterine growth restriction, aberrant regulation of components of the PPAR system parallels dysregulation of metabolism, inflammation and angiogenesis. This review summarizes current knowledge on the role of PPARs in regulating human trophoblast invasion, early placental development, and also in the physiology of clinical pregnancy and its complications. As increasingly indicated in the literature, pregnancy disorders, such as preeclampsia and gestational diabetes, represent potential targets for treatment with PPAR ligands. With the advent of more specific PPAR agonists that exhibit efficacy in ameliorating metabolic, inflammatory, and angiogenic disturbances, further studies of their application in pregnancy-related diseases are warranted.

Evolution of medical treatment for endometriosis: back to the roots?

Experimental evidence is accumulating to suggest that medicinal botanicals have anti-inflammatory and pain-alleviating properties and hold promise for treatment of endometriosis. Herein, we present a systematic review of clinical and experimental data on the use of medicinal herbs in the treatment of endometriosis. Although there is a general lack of evidence from clinical studies on the potential efficacy of medicinal herbs for the treatment of endometriosis-associated symptoms, our review highlights the anti-inflammatory and pain-alleviating mechanisms of action of herbal remedies. Medicinal herbs and their active components exhibit cytokine-suppressive, COX-2-inhibiting, antioxidant, sedative and pain-alleviating properties. Each of these mechanisms of action would be predicted to have salutary effects in endometriosis. Better understanding of the mechanisms of action, toxicity and herb-herb and herb-drug interactions permits the optimization of design and execution of complementary alternative medicine trials for endometriosis-associated pain. A potential benefit of herbal therapy is the likelihood of synergistic interactions within individual or combinations of plants. In this sense, phytotherapies may be analogous to nutraceuticals or whole food nutrition. We encourage the development of herbal analogues and establishment of special, simplified registration procedures for certain medicinal products, particularly herbal derivates with a long tradition of safe use.

A Western primer of Chinese herbal therapy in endometriosis and infertility.

Endometriosis is a disease that affects approximately 10% of all reproductive-aged women and the prevalence rises to 20-50% in infertile women. There is growing evidence that medicinal Chinese herbs with pain-alleviating and anti-inflammatory properties may be useful in the treatment of endometriosis and infertility, but the mechanisms of action of these herbs have yet to be investigated. In addition, studies of adequate design, sample size and appropriate control are lacking. Therefore, prospective randomized, controlled studies to evaluate the efficacy, mechanism of action and toxicities of Chinese herbs in the treatment endometriosis and infertility are needed.

Effects of phorbol dibutyrate on cell proliferation, apoptosis, and tumor necrosis factor-alpha expression in human endometrial adenocarcinoma cells.

OBJECTIVES: Recent evidence suggested that protein kinase C (PKC), a major cell cycle regulator in endometrial models, mimics progesterone withdrawal by inducing downstream signals. In the current study we examined the hypothesis that the PKC activator phorbol 12,13 dibutyrate (PDB) would inhibit cell proliferation and induce apoptosis in two endometrial adenocarcinoma cell (EAC) lines, HEC-1B and Ishikawa cells. We further examined whether the induction of tumor necrosis factor-alpha (TNF-alpha) might mediate these effects. METHODS: EAC lines were cultured under standard and serum-free conditions to study the effects of PDB on cell kinetics. Cell proliferation was determined by cell count using a hemacytometer and by incorporation of (3)H thymidine into 10% trichloracetic acid-precipitable DNA. Apoptosis was determined by measuring cytoplasmic histone-associated DNA fragments. Conditioned media concentrations of TNF-alpha were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). EACs were transfected with a -125-bp TNF-alpha promoter luciferase construct and treated with PDB to evaluate transcriptional activation. RESULTS: Activation of the PKC system with PDB (10 nM) decreased cell proliferation and mitogenesis in EACs. PDB induced apoptosis in both EAC lines. EACs exhibit basal TNF-alpha gene expression and protein secretion and these were increased potently by PDB. However, neutralization of TNF-alpha by addition of anti-TNF-alpha antibodies did not prevent the suppression of mitogenesis, induction of apoptosis, or activation of TNF-alpha gene expression by PDB. CONCLUSION: Activation of the PKC system leads to inhibition of cell proliferation, induction of apoptosis, and TNF-alpha expression in EACs. However, apoptosis in this setting does not appear to require TNF-alpha action. EACs provide an informative model to investigate aspects of endometrial epithelial remodeling that may occur under physiologic conditions of progesterone withdrawal.

Expression and regulation of CCR1 in peritoneal macrophages from women with and without endometriosis.

CCR1 is a CC chemokine receptor with high affinity for RANTES (regulated upon activation, normal T cells expressed and secreted). CCR1 protein and mRNA concentrations in native peritoneal cells were twofold greater, in cultured peritoneal cells threefold greater, in patients with endometriosis compared to patients without endometriosis, as determined by Western blotting fluorescence activated cell sorting analysis, reverse transcription-polymerase chain reaction, and in situ hybridization.

Estrogen receptor beta and matrix metalloproteinase 1 are coexpressed in uterine endometrium and endometriotic lesions of patients with endometriosis.

OBJECTIVE: To evaluate the local matrix metalloproteinase 1 (MMP-1), estrogen receptor (ER) alpha, and ERbeta protein expression in eutopic and dystopic tissue from patients with endometriosis and to compare the endometrial expression pattern in women with and without endometriosis. DESIGN: Immunohistochemical analysis of MMP-1, ERalpha, and ERbeta in paired samples of uterine and endometriotic endometrium from cycling women with endometriosis and in endometrial tissue from 37 healthy women. SETTING: Research laboratory at a medical school. PATIENT(S): Thirty-seven matched samples from endometriotic and corresponding endometrial biopsies obtained during the proliferative and secretory phase. Thirty-seven endometrial biopsies obtained from healthy women during comparable cycle phases. INTERVENTION(S): Sampling of endometrial and endometriotic tissue. MAIN OUTCOME MEASURE(S): Matrix metalloproteinase 1, ERalpha, and ERbeta protein expression in paraffin-embedded tissue biopsies was measured by IHC. RESULT(S): In patients with endometriosis, epithelial and stromal cells from endometriotic lesions both express significantly higher levels of MMP-1 and lower levels of ERalpha than corresponding cells in uterine endometrium. Endometriotic epithelium also expresses higher levels of ERbeta than of ERalpha. In both endometrial glands and corresponding endometriotic epithelium, the distribution of MMP-1 is correlated with ERbeta. No significant differences in endometrial ERalpha, ERbeta, or MMP-1 expression could, however, be detected when patients with endometriosis and healthy controls were compared. CONCLUSION(S): We have shown that MMP-1 and ERbeta are coexpressed and up-regulated in endometriotic lesions, whereas local ERalpha expression is down-regulated. The altered ERbeta/ERalpha ratio in endometriotic glands suggests that estrogenic effects on MMP-1 are primarily mediated via ERbeta, and the local control of MMP-1 in eutopic endometrium is not different from that observed in healthy cycling women.

Endometriosis: a genetic disease.

Endometriosis is a multifactorial disease affecting up to 15% of women of reproductive age. This condition is characterized by the presence and growth of endometrial cells outside the uterus. Susceptibility to endometriosis depends on complex interactions of immunologic, hormonal, environmental and genetic factors. Although familial inheritance plays a role, multiple candidate genes appear to be involved. New studies investigating the influence of genetic variants on endometriosis are published with increasing frequency. A number of technologies have emerged to facilitate progress in this field, including subtractive cDNA hybridization to identify secretory endometrial genes, DNA chip technology, differential display polymerase chain reaction, cytogenetics evaluation of endometriotic cells and tissues, and complementary methods in proteomics and informatics. One general approach to uncovering genes pivotal to endometriosis is to search systematically for perturbations in selective candidate genes or chromosomal regions using polymerase chain reaction. We describe here novel association studies on obvious candidates, including genes governing cancer susceptibility, hormone sensitivity or immunology. The assessment of mutations and polymorphisms may allow individualization of therapies as well as primary and secondary prevention strategies for endometriosis, aimed at high-risk populations.

Catechol-O-methyltransferase polymorphism and endometriosis.

PURPOSE: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy and 4-hydroxy catechols, which have been implicated in the pathogenesis of endometriosis. A COMT valine to methionine polymorphism (G-to-A) in exon 4 of the COMT gene is polymorphic in the human population, with 25% of Caucasians being homozygous for the low-activity allele (COMT-L) of the enzyme. In a case-control study we investigated whether this COMT polymorphism is associated with endometriosis. METHODS: Polymerase chain reaction was performed to analyze the COMT genotype among women with surgically and histologically confirmed endometriosis (study group; n = 91) and in women without evidence of endometriosis confirmed by laparoscopy or laparotomy (control group; n = 92). RESULTS: Allele frequencies for the low-activity allele (COMT-L) among women with endometriosis and controls were 0.50 and 0.50, respectively (p = 0.999; odds ratio = 1.0, 95% CI: 0.66-1.51). CONCLUSIONS: Our results suggest that the valine to methionine polymorphism in exon 4 of the COMT gene is not associated with the risk of endometriosis compared to a surgical control population.

Interleukin-1 receptor antagonist polymorphism in women with peritoneal adhesions.

Interleukin (IL)-1 has been shown to induce peritoneal adhesions. We determined the IL-1 receptor antagonist (IL-1RN) genotype with respect to the two most common variant alleles IL-1RN*2 and IL-1RN*3 in Caucasian women with peritoneal adhesions. One hundred seven women with surgically verified peritoneal adhesions and 79 controls without peritoneal adhesions served as controls. Univariate analysis showed an increased risk for peritoneal adhesions for Caucasian women carrying the mutant IL-1RN*2 allele (OR: 2.1; 95% CI: 1.3-3.4; P = 0.004). Multiple logistic regression analysis demonstrated an increased risk for peritoneal adhesions, which is independent of previous abdominal surgery and endometriosis. Our data suggest that IL-1RN*2 allele carriers have an increased risk for adhesion formation.