The biological effects of macrophage-colony-stimulating factor induced by progestin on growth and differentiation of endometrial adenocarcinoma cells.

Progestins have biological effects of regression and differentiation on human endometrial adenocarcinoma. We investigated the effects of progestin on the induction of macrophage-colony-stimulating factor (M-CSF) and its receptor messenger RNAs in the human endometrial adenocarcinoma cell line Ishikawa which has receptors for both estrogen and progesterone. Poly(A)+RNA extracted from Ishikawa cells cultured with or without synthetic progestin R5020 was subjected to Northern blot hybridization using M-CSF and c-fms cDNA probes. The expression of M-CSF mRNA in Ishikawa cells increased about 2.3 times following treatment with R5020 at 10(-7) M. Induction of M-CSF mRNA by R5020 was antagonized by anti-progestin RU486 in a dose-dependent manner. However, c-fms mRNA, coding the M-CSF receptor, was expressed constitutively in Ishikawa cells and its expression was not affected by hormonal treatment. We further examined the biological effects of M-CSF on endometrial cancer cells. Colony formation of Ishikawa cells in soft agar, which represents anchorage-independent cell growth, was inhibited by M-CSF treatment. On the other hand, accumulation of glycogen granules in cytoplasm detected by periodicacid-Schiff staining was observed in Ishikawa cells treated with M-CSF. These results indicate that M-CSF, whose gene expression was enhanced by progestin, suppressed growth and induced differentiation of endometrial adenocarcinoma cells. These effects of M-CSF on endometrial cancer cells are similar to those of progestins, so the effects of progestins on these cells are, at least in part, probably mediated by M-CSF in an autocrine or paracrine manner.     

A novel angiogenic role for prostaglandin F2alpha-FP receptor interaction in human endometrial adenocarcinomas

Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We investigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with FP receptor cDNA in the sense or antisense orientations. Treatment of Ishikawa cells with prostaglandin F2alpha (PGF2alpha) rapidly induced transphosphorylation of the epidermal growth factor receptor (EGFR) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 via the FP receptor. Activation of EGFR-Ras-mitogen-activated protein kinase/ERK kinase (MEK) signaling via the FP receptor resulted in an increase in VEGF promoter activity, expression of VEGF mRNA, and secretion of VEGF protein. These effects of PGF2alpha on the FP receptor could be abolished by treatment of cells with a specific FP receptor antagonist, chemical inhibitors of c-Src, matrix metalloproteinase, and EGFR kinase or by inactivation of signaling with dominant-negative mutant isoforms of EGFR, Ras, or MEK or with small inhibitory RNA oligonucleotides targeted against the EGFR. Finally, we confirmed that PGF2alpha could potentiate angiogenesis in endometrial adenocarcinoma explants by transactivation of the EGFR and induction of VEGF mRNA expression.     

Expression and function of activin receptors in human endometrial adenocarcinoma cells

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.     

Interleukin 11 regulates endometrial cancer cell adhesion and migration via STAT3

Endometrial carcinoma is the most common gynaecological malignancy. There is however a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Interleukin (IL) 11 is a pleiotropic cytokine that has a role in a number of cancers including colon and breast cancer. IL11 was recently found to be upregulated in endometrial cancers, however the function of IL11 in endometrial cancer is not known. This study aimed to determine the effects of IL11 on endometrial cancer cell proliferation, adhesion and migration. Three endometrial cancer cell lines, Ishikawa, HEC-1A and AN3CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of IL11 on endometrial cancer cell function. Cell proliferation and viability were assessed by BrdU and Wst-1 assays. Cell adhesion to the extracellular matrix proteins fibronectin, collagen I and IV, vitronectin and laminin was assessed. Modified boyden chambers were utilized to access IL11 action on migration and invasion, respectively. The specific effect of IL11 action on these processes was determined using a unique IL11 inhibitor. IL11 phosphorylated (p)-STAT3 protein abundance in all 3 cell lines but had no effect on pERK and pAKT abundance. Similarly, IL11 had no effect on cell proliferation and viability but increased adhesion of ANC3A cells to fibronectin while having no effect on the other extracellular matrix proteins. IL11 did not alter the adhesive properties of the Ishikawa and HEC-1A cells. In the AN3CA cells, IL11 treatment resulted in a 50% increase in migration and co-treatment with the specific IL11 inhibitor or a STAT3 inhibitor abolished the effect. This study shows a role for IL11 in endometrial cancer and suggests IL11 may be involved in endometrial cancer development and thus may be useful as a therapeutic target.     

Transforming growth factor gene expression in human endometrial adenocarcinoma cells: regulation by progestins.

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.     

The activation of an extracellular signal-regulated kinase by oestradiol interferes with the effects of trastuzumab on HER2 signalling in endometrial adenocarcinoma cell lines

Cellular response to oestradiol stimuli is mediated both by oestrogen receptor (ER) binding to oestrogen response elements (EREs) and by non-nuclear actions like activation of mitogen-activated protein kinase (MAPK) signal transduction. Therefore, oestradiol stimuli might be able to interfere with the action of antitumoral substances directed against receptor tyrosine kinase signalling. We investigated the effect of oestradiol on the inhibition of HER2 signalling by trastuzumab (Herceptin) in two human endometrial adenocarcinoma cell lines. Activation of the extracellular signal-regulated kinase (ERK-1/2), a major mediator of HER2 signalling, was measured by means of western blotting experiments and ERE activation was determined in transient reporter-gene assays. In endometrial Ishikawa and HEC-1A adenocarcinoma cells, HER2 signalling was inhibited by trastuzumab only in the absence of oestradiol. We were able to demonstrate that oestradiol counteracted the inhibitory effects of trastuzumab by rapid phosphorylation of ERK-1/2, a kinase downstream of the HER2 receptor. The pure anti-oestrogen ICI 182,780 was able to restore both the trastuzumab-triggered inhibition of the ERK-1/2 pathway and the antiproliferative action of this substance in Ishikawa cells. Our data suggest that combinations of trastuzumab with anti-oestrogens may be effective in the treatment of endometrial cancers with a positive ER and HER2 receptor status.     

细胞外信号调节激酶1/2在瘦素促进子宫内膜癌Ishikawa细胞增殖中的作用

背景与目的:流行病学研究提示瘦素与子宫内膜癌的发生有关,但其作用机制尚不清楚.瘦素作为促有丝分裂原,能够显著促进多种细胞的生长增殖.本研究的目的在于探讨细胞外信号调节激酶1/2(extraeellular signal-regulated kinase 1/2,ERK1/2)在瘦素促进子宫内膜癌细胞增殖中的作用.方法:免疫荧光染色检测Ishikawa细胞中瘦素受体的表达;于Ishikawa细胞中分别加入不同浓度的瘦素(0、10、50、100、150 ng/ml),作用不同时间(6、12、24h),MIT法检测各组细胞的增殖情况:同时应用ERK1/2激酶特异性抑制剂PD98059阻断ERK1/2磷酸化,观察其对瘦素促进Ishikawa细胞增殖的影响;应用免疫印迹技术检测100 ng/ml瘦素作用于Ishikawa细胞不同时间后(20、40、60 min)ERK1/2的活化水平(以P-ERK1/2与ERK1/2的比值表示).结果:免疫荧光检测结果证实Ishikawa细胞存在瘦素受体的表达:瘦素能明显促进Ishikawa细胞的增殖.在0～100 ng/ml范围内瘦素浓度越高.细胞增殖越显著,100 ng/ml瘦素作用24h其增殖效应最大(A值=0.73±0.02),100 ng/ml组与150 ng/ml组差异无统计学意义(P=0.129);PD98059能明显抑制瘦素对Ishikawa细胞的增殖作用.100 ng/ml瘦素和100μmol/L PD98059作用24h后,细胞增殖率为(6.88±0.86)%;Ishikawa细胞经100 ng/ml瘦素处理后,ERK1/2活化程度明显增高.结论:瘦素可能通过激活ERK1/2信号转导途径促进子宫内膜癌细胞的增殖.     

G蛋白偶联受体30在子宫内膜癌Ishikawa细胞中的表达及亚细胞定位

目的:探讨G蛋白偶联受体30(Gprotein-coupled receptor 30,GPR30)在人子宫内膜癌细胞株Ishikawa中的表达及亚细胞定位。方法:应用RT-PCR、蛋白质印迹法和免疫荧光技术分析Ishikawa细胞中GPR30蛋白的表达,FCM检测GPR30蛋白在细胞膜和细胞质中的阳性表达率,胶体金标记GPR30后在透射电子显微镜下观察GPR30蛋白的亚细胞定位。结果:Ishikawa细胞中有GPR30 mRNA和蛋白的表达,主要位于细胞膜和细胞质;细胞质中GPR30蛋白的阳性表达率[(20.10±0.13)%]显著高于细胞膜[(8.54±0.17)%],差异有统计学意义(P<0.01);透射电子显微镜下可见GPR30蛋白的亚细胞定位主要在细胞质的管网状网络上,可能是粗面内质网。结论:Ishikawa细胞中GPR30蛋白在细胞质中的表达高于细胞膜,其亚细胞定位主要位于粗面内质网,提示GPR30可能在子宫内膜癌中发挥重要作用。     

基于转录组测序分析骨髓间充质干细胞对子宫内膜癌细胞的影响

目的 探索骨髓间充质干细胞（bone mesenchymal stem cells,BMSCs）对子宫内膜癌Ishikawa细胞转录水平的影响。方法 用Lenti-EGFP慢病毒感染Ishikawa细胞,分别将表达增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）的Ishikawa细胞单独培养、与BMSCs接触共培养;通过流式细胞术分离Ishikawa细胞,并进行转录组测序分析筛选差异表达的mRNA和miRNA,通过miRWalk预测差异miRNA靶基因,对两者的交集基因进行GO、KEGG、蛋白网络互作分析;采用qRT-PCR和Western blot验证测序结果的准确性。结果 标记EGFP的Ishikawa细胞与BMSCs共培养后,分离出的Ishikawa-EGFP阳性细胞占33.6%,阴性细胞占10.5%。测序后共筛选出5 928个差异表达mRNA和111个差异表达miRNA。蛋白网络互作分析显示交集基因表达的蛋白之间相互作用,核心节点包括CDKN1A、JAK1、COL1A1、VCAN等。miRNA-mRNA网络图显示CDKN1A与...     

Differential effects of raloxifene and tamoxifen on the expression of estrogen receptors and antigen Ki-67 in human endometrial adenocarcinoma cell line

Tamoxifen and raloxifene are widely used in clinical practice. It has been found that tamoxifen treatment increases the risk of development of endometrial cancer. The effects of tamoxifen and raloxifene on endometrium might be caused by different estrogen receptor expression. The aim of the present study was immunohistochemical evaluation of the effects of tamoxifen and raloxifene on estrogen receptors, and Ki-67 antigen expression in the human endometrial adenocarcinoma Ishikawa cell line. Tamoxifen in concentrations of 10 microM and 20 microM increased ERalpha expression without any effect on ERbeta. All used concentrations of tamoxifen and raloxifene (0.1 nM, 1 nM, 10 nM, 1 micro M, 10 microM and 20 microM) had no effect on expression of ERbeta. Tamoxifen, but not raloxifene, increased Ki-67 antigen expression in the Ishikawa cell line. Tamoxifen, in contrast to raloxifene, increased proliferation of endometrial adenocarcinoma cells as well as exerted the shift of ERalpha/ERbeta ratio. Thus, it could be responsible for increased carcinogenic effect during tamoxifen treatment.     

ERRγ对子宫内膜癌细胞移植瘤组织中Akt、ERK表达的影响

目的:以ERα（+）子宫内膜癌细胞株Ishikawa为研究对象,建立子宫内膜癌细胞的裸鼠皮下移植瘤模型,进一步研究在子宫内膜癌细胞移植瘤中ERK、p-ERK、Akt、p-Akt蛋白的表达及其意义。方法:利用转染前后的Ishikawa细胞建立子宫内膜癌细胞裸鼠皮下移植瘤模型,采用免疫组织化学链霉亲和素-生物素过氧化物酶复合物(SABC法)检测裸鼠移植瘤中ERRγ蛋白的表达情况。Western blot方法检测移植瘤组织中Akt、p-Akt、ERK、p-ERK蛋白的表达情况。结果:免疫组化检测Ishikawa 细胞移植瘤组与no-silence Ishikawa细胞移植瘤组（空载体组）ERRγ蛋白表达阳性率无显著性差异（P＞0.05）,Ishikawa 细胞移植瘤组与ERRγ-shRNA Ishikawa细胞移植瘤组阳性率具有显著性差异（P＜0.05）。Western blot方法检测3组组织的活化情况:ERRγ-shRNA Ishikawa细胞移殖瘤组中Akt、p-Akt、ERK、p-ERK表达量均低于Ishikawa 细胞移殖瘤及no-silence Ishikawa细胞移殖瘤组（P＜0.05）。结论:沉默ERRγ基因的瘤组织的ERK及Akt的表达呈现低表达,从而可以推测:通过ERRγ的表达来调节ERK及Akt信号通路的表达,进一步抑制子宫内膜癌Ishikawa细胞增殖,促进其凋亡。     

Possible role of placental leucine aminopeptidase in the antiproliferative effect of oxytocin in human endometrial adenocarcinoma.

PURPOSE: Oxytocin (OT) was reported to inhibit the proliferation of various neoplastic tissues and cells, however, the regulation system remains unclear. This study examined the role of OT and its regulatory ability in endometrial adenocarcinoma. EXPERIMENTAL DESIGN: To investigate the possible function of placental leucine aminopeptidase (P-LAP) in endometrial adenocarcinoma, we transfected P-LAP cDNA into A-MEC cells, showing the lowest enzyme activity of P-LAP. Also we examined P-LAP protein expression in human endometrial adenocarcinoma. RESULTS: We demonstrated the presence of P-LAP, which is identical to cystine aminopeptidase as oxytocinase, in human endometrial adenocarcinoma tissues and found that the expression of P-LAP increase with advances in the grade. Exposure of endometrial adenocarcinoma cell lines to OT caused dose- and time-dependent inhibition of growth. Treatment with 10(-7) M OT for 72 h reduced cell growth by 62, 25, and 30% in A-MEC, HEC1A, and Ishikawa cells, respectively. P-LAP-transfectant cells not only partially recovered from OT-induced growth inhibition but also showed a higher growth rate than parental cells under condition without OT. An OT receptor antagonist and a protein kinase A inhibitor blocked OT-induced growth inhibition in A-MEC and A-MEC-pc cells but not in A-MEC-LAP cells. CONCLUSIONS: These findings suggested that P-LAP might be functionally positive on carcinoma cell growth by degrading suppressive peptides such as OT.     

HOXA10 基因在子宫内膜癌组织中的表达及其生物学作用

目的:探讨同源框A10（HOXA10）基因在子宫内膜癌组织中的表达及其对子宫内膜癌Ishikawa 细胞株凋亡、迁移及侵袭的影响。方法：收集2012 年至2013 年在鼓楼医院妇产科子宫内膜癌组织标本21 例、正常增殖期子宫内膜组织标本25 例,采用实时荧光定量PCR(qRT-PCR)及Western blotting 方法检测HOXA10 在子宫内膜癌组织及正常子宫内膜组织中的表达。将感染复数为5、10、20 MOI的ad-flag-HOXA10 腺病毒质粒及20 MOI的ad-flag-lacz 腺病毒质粒(对照组)感染人子宫内膜癌Ishikawa 细胞株,流式细胞术检测各组细胞凋亡情况。将50 nmol/L的si-HOXA10 及si-NC质粒转染Ishikawa 细胞株,分别为下调组及下调对照组;将20 MOI的ad-flag-HOXA10 及20 MOI的ad-flag-lacz 腺病毒质粒感染Ishikawa 细胞株,分别为上调组及上调对照组;Transwell 小室检测各组细胞迁移及侵袭能力。结果：在子宫内膜癌组织中HOXA10 mRNA 表达量比正常子宫内膜组织中显著降低[ （0.56±0.14）vs (1.36±0.33),P<0.01],其蛋白表达量同样显著降低[ （1.01±0.25）vs (2.10±0.71),P<0.01]。上调HOXA10后5、10、20 MOI 组细胞的凋亡率明显升高,且大多数处于早期凋亡[ （50.92±8.79）%、(55.17±4.07)%、(76.10±3.65)% vs (7.74±0.15)%,均P<0.01]。下调HOXA10 表达后迁移细胞数显著增加[ （248±25）vs (135±15)个,P<0.01],上调HOXA10 表达后迁移细胞数显著减少[（50±6）vs (100±13) 个,P<0.01];下调HOXA10 表达后侵袭细胞数显著增多[ （131±18）vs (66±9)个,P<0.01],上调HOXA10 表达后侵袭细胞数显著减少[ （34±8）vs（60±4）个,P<0.01]。结论：HOXA10 基因在子宫内膜癌内膜中表达低于正常内膜,子宫内膜癌Ishikawa细胞株中上调HOXA10 基因表达能促进细胞凋亡并抑制其迁移和侵袭能力。     

Expression of keratinocyte growth factor and its receptor in human endometrial cancer in cooperation with steroid hormones

The keratinocyte factor (KGF) and its receptor (KGFR) are implicated in tissue development and repair. We studied the expression and functions of KGF and KGFR in association with estrogen and progesterone in human endometrial tissues and cells. In non-cancerous human endometrial tissues in the secretory phase, a strong immunoreactivity of KGF in glands, stromal cells, and smooth muscle cells of spiral arteries was detected; however, in proliferative-phase tissues, the immunoreactivity of KGF or KGFR was weak or absent. Most of the 32 endometrioid adenocarcinoma cases showed positive KGF and KGFR stainings (90.6 and 71.9%, respectively). We then studied, using Ishikawa well-differentiated human endometrial cancer cell line that expresses estrogen receptor (ER) and progesterone receptor (PR), the expression of KGF and KGFR in conjunction with estrogen and progesterone, and observed that the KGFR expression of Ishikawa cells was upregulated by estrogen and that this upregulation was markedly enhanced by the coadministration of progesterone. We also observed that KGF administration to cells, with KGFR upregulated expression, stimulated ERK1/2 phosphorylation and cell adhesion to fibronectin. The implications of the hormone-stimulated KGF-KGFR expressions in the regulation of cell behavior associated with human endometrial cancer are discussed.     

Differential effects of estrogen and antiestrogen on transforming growth factor gene expression in endometrial adenocarcinoma cells.

While antiestrogens are useful agents in the treatment of breast cancer, the usefulness of these agents in the treatment of endometrial cancer remains controversial. There is some concern that the currently available antiestrogens may have partial agonist activity in uterine tissue. To better understand the mechanisms by which estrogens and antiestrogens modulate growth of endometrial adenocarcinoma cells, we have compared the effects of 17-beta estradiol and three antiestrogens, 4-hydroxytamoxifen (OH-TAM), ICI 164384, and LY 117018 on proliferation and transforming growth factor (TGF) mRNA accumulation in two human endometrial adenocarcinoma cell lines. In HEC-50 cells, neither estradiol nor anti-estrogens had any effect on cell proliferation or TGF mRNA abundance under estrogen-depleted culture conditions [basal medium containing 1% twice charcoal-treated fetal bovine serum (ctFBS)] or in the presence of estrogen (basal medium containing 5% fetal bovine serum). At very high concentrations, both estradiol and OH-TAM caused a small decrease in HEC-50 cell proliferation in medium containing 5% serum. In contrast, the antiestrogens had different effects on Ishikawa cells, depending upon the culture conditions. In medium containing 5% fetal bovine serum, the antiestrogens inhibited cell proliferation and significantly decreased TGF-alpha mRNA abundance and TGF-alpha secretion. OH-TAM was more potent than the other antiestrogens. Under these culture conditions, estradiol had no effect on cell proliferation or TGF-alpha mRNA levels but increased TGF-alpha secretion. In medium supplemented with 1% ctFBS, estradiol increased cell proliferation and TGF-alpha mRNA (2.72-fold, P less than 0.005) and TGF-alpha secretion (700 +/- 156 versus 250 +/- 23 pg/10(6) cells/24 h, P less than 0.05), whereas OH-TAM, which also stimulated cell proliferation, reduced TGF-alpha mRNA abundance (P less than 0.05) but had no significant effect on TGF-alpha secretion. Under these conditions, ICI 164384 and LY 117018 had no effect on either cell proliferation or TGF-alpha expression. Estradiol treatment decreased, whereas OH-TAM increased, epidermal growth factor receptors in Ishikawa cells. Both estradiol and the antiestrogens decreased TGF-beta 1 mRNA abundance when cells were grown in media containing 1% ctFBS. In summary, the response of human endometrial adenocarcinoma cells to estrogen and antiestrogens varied between cell lines and was dependent upon the culture conditions used. In addition, OH-TAM, unlike the other two antiestrogens tested, had growth-stimulating effects on Ishikawa cells.     

LAT1 is a putative therapeutic target in endometrioid endometrial carcinoma

l ‐type amino acid transporters (LAT1‐4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1‐4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease‐free survival. The pan‐LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.     

In 2D and 3D Cell Culture Models,Effects of Endothelial Cells on E-cadherin / β-catenin Expression Levels and Spheroid Sizes in Ishikawa Cells

Objective: Increasing evidence shows that three dimensional cell culture models better reflect the in vivo tumor microenvironment than two dimensional cell culture models. Co-culture models are ideal cell culture models for understanding the communication between cells and the in vivo microenvironment. Changes in expression levels of E-cadherin are closely related to cancer metastasis and progression. β-catenin mediates cell adhesion of E-cadherin. Endothelial cells are stromal cell components in the tumor microenvironment. It is known that there is little or no expression of E-cadherin in endothelial cells. Methods: In our study, both two-dimensional and three dimensional mono-culture and co-culture models were created using Huvec and Ishikawa cells (endometrial cancer cell lines) to better reflect cell interactions. Spheroids were followed for three days in the three-dimensional cell culture model. E-cadherin and β-catenin expression levels of two-dimensional and three dimensional mono-culture and co-culture models were measured by immunofluorescence staining. Spheroid images were recorded using a Z-stack. Intensity measurements in both two-dimensional and three-dimensional mono-culture and co-culture models were made using the Image J software. Study groups were evaluated by one-way analysis of variance (One-Way ANOVA). Values of p <0.05 were considered statistically significant. Results: The size of the co-culture spheroids was recorded significantly larger than the mono-culture spheroids (p <0.0001). In two (p = 0.0175) and three dimensional models (p <0.0001), expression levels of E-cadherin in the mono-culture of Ishikawa cells were recorded significantly higher than in Huvec and co-culture cells. Likewise, while the expression levels of β-catenin were higher in the mono-culture of Ishikawa cells in two-dimensional models (p <0.05), no significant difference was observed in three dimensional models (p> 0.05). Conclusion: In summary, it has been noted that the expression levels of E-cadherin are significantly reduced in co-cultures of Ishikawa cells with Huvec cells in both two and three dimensions . These results support the idea that endothelial cells may cause changes in endometrial cancer progression by suppressing E-cadherin expression in Ishikawa cells.