Progesterone-mediated regulation of catechol-O-methyl transferase expression in endometrial cancer cells

The effects of estrogen and progesterone on the expression of estrogen-metabolizing enzymes such as catechol-O-methyl transferase (COMT) are not known. COMT converts genotoxic catecholestrogens to anticarcinogenic methoxyestrogens in the endometrium. The aim of this study is to investigate the effect of progesterone on COMT expression in well-differentiated endometrial cancer cells. The wild-type Ishikawa cell line as well as progesterone receptor A- or progesterone receptor B-transfected Ishikawa cells were used for in vitro studies. The regulation of COMT expression by progesterone was studied using Western blots, Hoechst dye DNA proliferation studies, and wild-type and/or site-directed mutagenesis of COMT promoter 1-luciferase reporter gene. Progesterone upregulated COMT protein expression in Ishikawa cells through progesterone receptor A isoform. COMT promoter activity was differentially regulated by the 3 half-site progesterone response elements in the COMT promoter. High doses of 2-ME2 inhibited Ishikawa cell proliferation. These data suggest that COMT expression is hormonally regulated in well-differentiated human endometrial cancer cells. COMT regulation and 2-ME2 production in the endometrium may affect endometrial carcinogenesis.     

Distinct functional differences of human progesterone receptors A and B on gene expression and growth regulation in two endometrial carcinoma cell lines

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59).Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells.We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.     

雌激素受体相关受体α过度表达对雌激素受体阴性的子宫内膜癌细胞增殖的影响

目的探讨雌激素受体相关受体α（ERRα）在雌激素受体（ER）阴性及阳性的子宫内膜癌细胞中的作用。方法 将真核表达质粒pSG—ERRα（0．5、1．0、1．5、2．5μg）瞬时转染子宫内膜癌细胞株HEC-1A（ER阴性）、HEC-1B（ER阴性）、Ishikawa（ER阳性）,采用定量RT-PCR技术和蛋白印迹法（westernblot）检测ERRα mRNA和蛋白的表达情况;采用流式细胞仪分析细胞周期,并计数细胞的增殖情况。结果 转染pSG—ERRα质粒后,HEC-1A、HEC-1B、Ishikawa细胞在mRNA和蛋白水平均能检测到ERRet的表达增加。HEC-1B、HEC-1A、Ishikawa细胞未转染时ERRα mRNA的表达水平分别为2104．2、2870．6、1476．8copies／ng,转染后3者ERRα mRNA的表达水平分别为9835．3、9644．4、8008．6copies／ng,分别与各自未转染的细胞比较,差异均有统计学意义（P值分别为0．004、0．002、0．002）。HEC-1A、HEC-1B、Ishikawa细胞未转染时ERRα蛋白的表达水平分别为0．823、0．192、0．673,转染后3者ERRα蛋白的表达水平分别为1．128、1．104、1．008,分别与各自未转染的细胞比较,差异均有统计学意义（P〈0．05）。随着转染pSG—ERRα质粒质量的增加,HEC-1A、HEC-1B细胞的S期和G2／M期细胞比例明显上升（P〈0．01）。HEC-1B、HEC-1A细胞转染0．5、1．0μg pSG-ERRα质粒后,细胞在转染后24—96h间生长速度显著加快（P〈0．05）。结论ERRα过度表达是ER阴性的子宫内膜癌细胞株HEC-1A、HEC-1B的一种细胞增殖机制。     

Oviductal glycoprotein 1 (OVGP1) is expressed by endometrial epithelium that regulates receptivity and trophoblast adhesion

Purpose

To study the regulation and functions of oviductal glycoprotein 1 (OVGP1) in endometrial epithelial cells.

Methods

Expression of OVGP1 in mouse endometrium during pregnancy and in the endometrial epithelial cell line (Ishikawa) was studied by immunofluorescence, Western blotting, and RT-PCR. Regulation of OVGP1 in response to ovarian steroids and human chorionic gonadotropin (hCG) was studied by real-time RT-PCR. OVGP1 expression was knockdown in Ishikawa cells by shRNA, and expression of receptivity associated genes was studied by real-time RT-PCR. Adhesion of trophoblast cell line (JAr) was studied by in vitro adhesion assays.

Results

OVGP1 was localized exclusively in the luminal epithelial cells of mouse endometrium at the time of embryo implantation. Along with estrogen and progesterone, hCG induced the expression of OVGP1 in Ishikawa cells. Knockdown of OVGP1 in Ishikawa cells reduced mRNA expression of ITGAV, ITGB3, ITGA5, HOXA10, LIF, and IL15; it increased the expression of HOXA11, MMP9, TIMP1, and TIMP3. Supernatants derived from OVGP1 knockdown Ishikawa cells reduced the adhesiveness of JAr cells in vitro. Expression of OVGP1 mRNA was found to be significantly lowered in the endometrium of women with recurrent implantation failure.

Conclusion

OVGP1 is specifically induced in the luminal epithelium at the time of embryo implantation where it regulates receptivity-related genes and aids in trophoblast adhesion.

     

EGFR过表达降低人子宫内膜癌细胞孕激素敏感性的研究

目的:检测表皮生长因子受体(EGFR)基因在子宫内膜癌孕激素敏感细胞株Ishikawa及孕激素不敏感细胞株KLE的表达,探讨EGFR基因过表达对人子宫内膜癌细胞孕激素敏感性的影响。方法:实时定量PCR法和蛋白印迹法检测Ishikawa和KLE细胞中EGFR及PR-BmRNA和蛋白的表达。将EGFR全长cDNA真核表达质粒在脂质体介导下转染至Ishikawa细胞,同时以转染空载体和未转染的Ishikawa细胞为对照,分别应用实时定量PCR检测各组细胞EGFR、PR-BmRNA表达的变化,应用蛋白印迹法检测各组细胞EGFR、PR-B蛋白表达的变化;CCK-8法观察转染EGFR基因后Ishikawa细胞对孕激素敏感性的变化。结果:(1)Ishikawa细胞中,EGFRmRNA和蛋白的表达明显低于KLE细胞(P<0.001),而PR-BmRNA和蛋白的表达则显著高于KLE细胞(P<0.001);(2)稳定转染EGFR基因后,Ishikawa细胞中EGFRmRNA和蛋白的表达水平明显高于转染空载体和未转染的Ishikawa细胞(P<0.001),而PR-BmRNA和蛋白的表达水平则显著降低;(3)10-8、10-7、10-6、10-5mol/L的MPA对未转染和转染空载体的Ishikawa细胞的抑制作用显著(P<0.05),但对稳定过表达EGFR的Ishikawa细胞无明显抑制作用(P>0.05)。结论:转染EGFR基因能有效提高Ishikawa细胞内EGFR基因的表达,但可下调PR-B基因的表达使Ishikawa细胞对MPA不敏感。     

Differential gene expression in progesterone-sensitive and progesterone-insensitive endometrial carcinoma cells

High doses of progesterone are used in the treatment of advanced and recurrent endometrial cancer. Unfortunately the response rate is relatively low: 10-30%. The mechanisms involved in the development of insensitivity to progesterone treatment of endometrial cancer tissue are largely unknown. As tumour development is thought to be associated with a cascade of genetic alterations, it can be expected that genetic changes are involved in the development of progesterone insensitivity in endometrial carcinomas. We therefore started an investigation to identify, isolate and characterise progesterone-regulated genes involved in progesterone-induced growth inhibition in endometrial carcinoma cells. Using differential display PCR eight progesterone-regulated cDNA clones were identified in endometrial carcinoma cell lines. Four of these progesterone-regulated cDNA clones were regulated in the for growth progesterone-sensitive cell line IK-3H12 and not regulated in the for growth-insensitive cell line ECC-1. This indicates that these four cDNA clones represent potentially important genes, which could be involved in inhibition of growth of endometrial carcinoma tissue by progesterone.     

Luteinizing hormone-releasing hormone agonist triptorelin and antagonist cetrorelix inhibit EGF-induced c-fos expression in human gynecological cancers

OBJECTIVES: Spontaneous and epidermal growth-factor-induced proliferation of human gynecological cancer cell lines is dose- and time-dependently reduced by treatment with the luteinizing hormone-releasing hormone (LHRH) agonist triptorelin and antagonist Cetrorelix. This antiproliferative activity is probably directly mediated through the LHRH receptors expressed by the tumor cells interacting with growth-factor-dependent mitogenic signal transduction. We have examined whether epidermal growth-factor (EGF)-induced expression of the early response gene c-fos is reduced by LHRH analogs. METHODS: Human endometrial (Ishikawa, Hec-1A), ovarian (EFO-21, EFO-27, SK-OV-3), and breast cancer cell lines (MCF-7) were rendered quiescent by incubation (72 h) in the absence of fetal calf serum and phenol red. This was followed by a 15-min incubation in the absence or presence of the LHRH agonist triptorelin (100 nM) or the antagonist Cetrorelix (100 nM) before the cells were stimulated for 10 min with EGF (100 nM). C-fos mRNA expression was determined by semi-quantitative RT-PCR using a synthetic DNA fragment as internal standard. C-Fos protein synthesis was determined by SDS-PAGE and semi-quantitative Western blotting. RESULTS: In cells derived from endometrial and ovarian cancer, maximal c-fos mRNA expression (seven- to ninefold over basal level) was obtained 30 min after EGF stimulation. In the breast cancer cell line MCF-7 this effect was obtained 60 min after EGF treatment. In all of the lines expressing LHRH receptor, EGF-induced c-fos mRNA expression as well as c-Fos protein synthesis was dose-dependently reduced by treatment with LHRH agonists and antagonists. At 100 nM concentrations of the LHRH analogs, c-fos expression was reduced to baseline levels. No effect of LHRH analogs on EGF-induced c-fos expression was observed in the ovarian cancer cell line SK-OV-3, which does not express the LHRH receptor. CONCLUSIONS: These results suggest that the binding of LHRH agonists and antagonists to their receptors inhibits the mitogenic signal transduction pathway of the EGF receptor in endometrial, ovarian, and breast cancer cell lines. The coupling of both signal transduction systems mediates the antiproliferative effect of LHRH analogs.     

Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Objectives

To examine the effects of combination therapy with metformin and paclitaxel in endometrial cancer cell lines.

Methods

ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed after exposure to paclitaxel and metformin. Cell cycle progression was assessed by flow cytometry. hTERT expression was determined by real-time RT-PCR. Western immunoblotting was performed to determine the effect of metformin/paclitaxel on the mTOR pathway.

Results

Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC₅₀ values of 1-5 nM and 5-10 nM for Ishikawa and ECC-1 cells, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with metformin (0.5 mM) resulted in a significant synergistic anti-proliferative effect in both cell lines (Combination Index < 1). Metformin induced G1 arrest in both cell lines. Paclitaxel alone or in combination with metformin resulted in predominantly G2 arrest. Metformin decreased hTERT mRNA expression while paclitaxel alone had no effect on telomerase activity. Metformin stimulated AMPK phosphorylation and decreased phosphorylation of the S6 protein. In contrast, paclitaxel inhibited AMPK phosphorylation in the ECC-1 cell line and induced phosphorylation of S6 in both cell lines. Treatment with metformin and paclitaxel resulted in decreased phosphorylation of S6 in both cell lines but only had an additive effect on AMPK phosphorylation in the ECC-1 cell line.

Conclusions

Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway. This combination may be a promising targeted therapy for endometrial cancer.     

子宫内膜癌Dickkopf1表达及对内膜癌侵袭力的影响

目的:研究Dickkopf1在子宫内膜癌组织和Ishikawa细胞系的表达及对子宫内膜癌侵袭力的影响。方法:应用免疫组化和免疫荧光法检测子宫内膜癌组织和Ish-ikawa细胞系中Dickkopf1的表达定位;向子宫内膜癌Ishikawa细胞系施加外源性Dickko-pf1,应用Transwellchamber法进行体外侵袭试验,检测它对内膜癌侵袭能力的影响。结果:Dickkopf1在子宫内膜癌腺上皮和基质组织中均有表达,腺上皮的表达高于基质;Dickkopf1主要表达于内膜癌细胞浆和细胞膜中;施加外源性Dickkopf1后,子宫内膜癌细胞的侵袭能力下降。结论:Dickkopf1能减弱子宫内膜癌的侵袭能力,它有望作为内膜癌的治疗靶点。     

雌激素及其阻滞剂对Ishikawa细胞中ETS-1和VEGF调控的研究

目的:探讨雌激素及其阻滞剂对子宫内膜癌细胞系Ishikawa细胞中转录因子ETS-1和血管内皮细胞生长因子(VEGF)表达的调控机制。方法:用RT-PCR法检测分别使用不同浓度雌激素、雌激素受体阻滞剂氟维司群处理后Ishikawa细胞中ETS-1基因表达水平的变化;用细胞爬片免疫组化法检测分别使用这两种不同浓度药物后Ishikawa细胞中ETS-1与VEGF表达的差别;采用ELISA法检测分别使用两种不同浓度的药物后Ishikawa细胞培养液中VEGF蛋白表达量的变化。结果:(1)随着雌激素浓度增加,ETS-1基因表达上调(FP=3.84,P<0.05);随着氟维司群浓度增加,ETS-1基因表达下调(FP=7.01,P<0.01);(2)随着雌激素浓度增加,ETS-1(FP=3.72,P<0.05)和VEGF(FP=3.68,P<0.05)蛋白质水平表达都增加;随着氟维司群浓度增加,ETS-1(FP=4.37,P<0.05)和VEGF(FP=4.09,P<0.05)蛋白质水平的表达都减少;(3)随着雌激素浓度增加,分泌型VEGF蛋白表达量上升(FP=3.91,P<0.05);随着氟维司群浓度增加,分泌型VEGF蛋白表达量下调(FP=7.34,P<0.01)。结论:雌激素可促进Ishikawa细胞中ETS-1和VEGF的表达,VEGF表达与ETS-1表达呈正相关。     

华蟾素对子宫内膜癌Ishikawa细胞RRM2表达的影响和意义

目的:探讨华蟾素对体外培养的子宫内膜癌Ishikawa细胞增殖、侵袭力及其对RRM2表达的影响和意义。方法:体外培养Ishikawa细胞,经不同浓度华蟾素干预后,用RT-PCR及Western blot分别检测RRM2在mRNA及蛋白水平上的表达,用MTT法检测处理前后细胞的增殖,transwell小室分析细胞体外的侵袭力。结果:华蟾素能显著减少RRM2在mRNA及蛋白水平的表达,有效地抑制Ishikawa细胞增殖,降低侵袭力,研究证实华蟾素终浓度3.0mg/ml是最佳抑制浓度,对照组Ishikawa细胞组则无上述效应。结论:华蟾素可明显降低Ishikawa细胞中RRM2表达,抑制细胞增殖,降低细胞侵袭力。     

Epidermal growth factor receptor signaling enhanced by long-term medroxyprogesterone acetate treatment in endometrial carcinoma

OBJECTIVE: Progestin is an effective endocrine treatment for patients with atypical hyperplasia or with endometrial carcinoma that is estrogen receptor (ER) positive and progesterone receptor (PR) positive. However, long-term progestin treatment may lead to resistance. We have studied the progestin resistance phenotype that frequently develops in endometrial carcinoma. METHODS: Ishikawa endometrial carcinoma cells were cultured for a long period (10 months) in the presence of the synthetic progestin medroxyprogesterone acetate (MPA), thereby generating a subline refractory to the growth-suppressive effects of MPA. RESULTS: The MPA-resistant subline showed growth stimulation rather than inhibition after MPA treatment. Immunocytochemical analysis showed reduced ER alpha and PR-B expression and increased ER beta expression in this subline compared with parental Ishikawa cells. Progestin-resistant Ishikawa cells also showed increased expression of transforming growth factor alpha (TGFalpha), the epidermal growth factor receptor (EGFR), and EGFR tyrosine kinase (EGFR-TK); MPA treatment further stimulated the expression of TGFalpha in these cells. Additionally, progestin-resistant Ishikawa cells were highly sensitive to growth stimulation by TGFalpha and to growth inhibition by the EGFR-TK-specific inhibitor AG1478, and they showed increased dependence on TGFalpha-EGFR signaling. CONCLUSIONS: Our results suggest that prolonged treatment of endometrial carcinoma cells with MPA induces resistance to the growth-suppressive effects of MPA and enhances cancer cell proliferation. The downregulation of ER alpha and PR-B, the upregulation of ER beta, and highly activated TGF-EGFR signaling are thus likely to contribute to progestin resistance in endometrial carcinoma. Therefore, an EGFR-TK-specific inhibitor might be useful in the treatment of progestin-resistant endometrial carcinoma.     

ING4在正常子宫内膜及子宫内膜癌的表达及临床意义

目的:研究生长抑制因子4(ING4)在正常子宫内膜及子宫内膜癌组织中的表达,探讨ING4在子宫内膜癌发生、发展中的作用,为子宫内膜癌的临床预防及治疗提供新的标志物。方法:利用荧光定量PCR、半定量RT-PCR、Western-blot及免疫组化等方法分别检测正常子宫内膜细胞及子宫内膜癌细胞系Ishikawa、HHUA及子宫内膜癌组织中ING4基因的表达,并进行分析比较。结果:子宫内膜癌细胞中ING4基因的表达量显著低于正常子宫内膜细胞(P<0.05),且ING4在Ishikawa和HHUA这两种子宫内膜癌细胞系之间的表达量没有显著性差异(P>0.05)。Western-blot结果显示,正常子宫内膜细胞的ING4蛋白表达量显著高于Ishikawa和HHUA这两种细胞系(P<0.05),HHUA细胞系的ING4蛋白表达量显著高于Ishikawa细胞系(P<0.05)。免疫组化结果显示,ING4在不同组织内的表达量差异有统计学意义(P<0.05),正常子宫内膜细胞内的表达量最高,其次是复杂性及不典型增生子宫内膜,子宫内膜癌内的ING4表达量最低。结论:ING4基因表达的下调与子宫内膜癌发生、发展可能有一定关系,对子宫内膜癌的诊断有一定的参考意义。     

miR-26a-5p通过靶向THAP2促进子宫内膜癌细胞凋亡

目的:研究miR-26a-5p靶向THAP2对子宫内膜癌细胞凋亡的影响。方法:实时荧光定量RT-PCR法检测子宫内膜癌细胞中miR-26a-5p表达水平。构建以慢病毒为载体的miR-26a-5p过表达和干扰质粒,分别转染子宫内膜癌细胞系Ishikawa与KLE,实时荧光定量RT-PCR法检测miR-26a-5p及下游分子THAP2转录水平。流式细胞技术分析miR-26a-5p表达对子宫内膜癌细胞凋亡的影响。结果:子宫内膜癌细胞Ishikawa与KLE均表达miR-26a-5p;miR-26a-5p过表达能增加THAP2的转录水平,促进子宫内膜癌细胞凋亡;miR-26a-5p下调能抑制THAP2的转录水平,抑制子宫内膜癌细胞凋亡。结论:miR-26a-5p可提高THAP2的转录水平,促进子宫内膜癌细胞凋亡,明确这种调控机制可能为子宫内膜癌预防诊断和治疗带来新的契机。