RL95-2 Endometrial Cancer Cell Research Articles

Platycodin D inhibits the proliferation, invasion and migration of endometrial cancer cells by blocking the PI3K/Akt signaling pathway via ADRA2A upregulation.

Endometrial cancer (EC) is a complex disease that affects the reproductive health of females worldwide. Platycodin D (PD) is known to exert numerous anticancer effects, markedly inhibiting cell proliferation, inducing apoptosis and causing cell cycle arrest in several types of cancer. The present study aimed to explore the mechanisms underlying the effects of PD in EC cells. The viability and proliferation of human endometrial stromal cells (ESCs) and RL95-2 EC cells following treatment with PD were evaluated using Cell Counting Kit-8, MTT and colony formation assays. Wound healing and Transwell assays were also performed to assess the migration and invasion of EC cells following treatment with PD. The expression levels of α2A-adrenergic receptor (ADRA2A) were measured using reverse transcription-quantitative PCR and western blotting assays with and without PD treatment and following transfection with short hairpin (sh) RNAs targeting ADRA2A2. Moreover, western blot analysis was performed to measure the expression levels of Ki67, PCNA, MMP2 and MMP9 and the phosphorylation of proteins of the PI3K/Akt signaling pathway. The results demonstrated that treatment with PD markedly decreased the proliferation, invasion and migration of EC cells, and reduced activation of the PI3K/Akt signaling pathway in EC cells. Moreover, transfection with sh-ADRA2A attenuated the effects of PD. ADRA2A expression was downregulated in EC cells compared with ESCs, and ADRA2A expression was elevated in EC cells following treatment with PD. In conclusion, the present study indicates that PD blocked the PI3K/Akt signaling pathway via the upregulation of ADRA2A expression, thereby inhibiting the proliferation, invasion and migration of EC cells.

M6A‐related long noncoding RNAs predict prognosis and indicate therapeutic response in endometrial carcinoma

N6-methyladenosine (m6A) has been identified as the most common, abundant, and conserved internal transcriptional modification. Long noncoding RNAs (lncRNAs) are noncoding RNAs consisting of more than 200 nucleotides, and the expression of various lncRNAs may affect cancer prognosis. The impact of m6A-associated lncRNAs on uterine corpus endometrial carcinoma (UCEC) prognosis is unknown. In this study, UCEC prognosis-related m6A lncRNAs were screened, bioinformatics analysis was performed, and experimental validation was conducted. Endometrial carcinoma (EC) and normal tissue samples were obtained from The Cancer Genome Atlas. The prognosis-related m6A lncRNAs screened by the least absolute shrinkage and selection operator method were used for multivariate Cox proportional risk regression modeling. Principal component analysis and Gene Ontology, immune function difference, and drug sensitivity analyses of the prognostic models were performed. Prognostic analysis was conducted for m6A-associated lncRNAs. The immune infiltration relationship of m6A-associated lncRNAs in EC was identified using the ssGSEA immune infiltration algorithm. A competing endogenouse RNA network was constructed using the LncACTdb database. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays were used to validate the differences in m6A-related lncRNA expression in normal and EC cells. CDKN2B-AS1 and MIR924HG were found to be risk factors for EC. RAB11B-AS1 was a protective factor in EC patients. MIR924HG expression was upregulated in KLE and RL95-2 endometrial cancer cell lines. Prognostic models involved RAB11B-AS1, LINC01812, HM13-IT1, TPM1-AS, SLC16A1-AS1, LINC01936, and CDKN2B-AS1. The high-risk group was more sensitive to five compounds (ABT.263, ABT.888, AP.24534, ATRA, and AZD.0530) than the low-risk group. These findings contribute to understanding of the function of m6A-related lncRNAs in UCEC and provide promising therapeutic strategies for UCEC.

17β-Estradiol promotes endometrial cancer proliferation and invasion through IL-6 pathway.

Accumulating evidence revealed that the leading risk factor of endometrial cancer is exposure to endogenous and exogenous estrogens, while the exact mechanism underlying estrogen contribution to endometrial cancer progression has not been elucidated clearly. Interleukin (IL)-6 has been verified to be critical for tumor progression in several human cancers. In this study, we provided evidence that 17β-estradiol (E2) could significantly promote endometrial cancer cells viability, migration and invasion through activation of IL-6 pathway, which involved in its downstream pathway and target genes (p-Stat3, Bcl-2, Mcl-1, cyclin D1 and MMP2). Meanwhile, utilization of IL-6-neutralizing antibody could partially attenuate the increased cancer growth and invasion abilities in Ishikawa and RL95-2 endometrial cancer cell lines and an orthotopic endometrial cancer model. We established a causative link between estrogen and IL-6 signaling activation in the development of endometrial cancer. The molecular mechanism defined in this study provided the evidence that E2 promotes endometrial carcinoma progression via activating the IL-6 pathway, indicating that interruption of IL-6 might be an essential therapeutic strategy in estrogen-dependent endometrial cancer.

Abstract 3335: Characterizing the MAXH28R secretome in endometrial cancer

Abstract We have previously shown that the recurrent endometrial cancer MAX p.His28Arg mutation (MAXH28R) is associated with a proangiogenesis phenotype in both in vitro and xenograft models. The purpose of this study is to characterize the MAXH28R secretome to identify the proangiogenic and other paracrine factors contributing to MAXH28R associated tumorigenesis. Bottom-up high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of secreted proteins coupled with microarray analysis of global gene expression was performed in AN3CA endometrial cancer cell lines that stably express the long isoform of MAX-wild type (MAXWT) or MAXH28R. Orthogonal validation of differentially expressed proteins was performed by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using MAXWT- or MAXH28R-expressing AN3CA cell lines. Bioinformatic analysis of the proteins secreted into conditioned media (secretome) identified 114 proteins differentially expressed by MAXH28R compared to MAXWT-expressing AN3CA cells (P < 0.05). Differential expression of proteins was highly correlated with mRNA expression (Pearson r = 0.75, P < 0.001), indicating that a subset of the differentially expressed proteins are potential direct transcriptional targets of MAX. Secretome analysis revealed that Laminin-β1 expression in MAXH28R was reduced more than 100-fold compared to MAXWT (P < 0.001), while ALCAM (CD166) expression was increased 3.5-fold (P < 0.001). Decreased expression of TGFβ1 (5.9-fold decrease, P = 0.023) and TGFβ2 (3-fold decrease, P = 0.009) was seen for MAXH28R cells compared to MAXWT. Differential expression of Laminin-β1 and ALCAM was confirmed by WB analysis and TGFβ1 and TGFβ2 by ELISA. Our analyses revealed extensive changes in the MAXH28R secretome, as exemplified above. Reduced Laminin-β1 highlights alteration in the extracellular matrix with implications for cell adhesion and integrin signaling. Increased ALCAM expression indicates changes to cell-cell adhesion. Elevated levels of ALCAM in conditioned media are consistent with increased ALCAM shedding, which is a marker of more aggressive tumors in a variety of malignancies. Reduced TGFβ ligand levels suggest potential alterations in paracrine and autocrine TGFβ signaling. However, the impact on canonical and noncanonical TGFβ signaling in MAX mutant endometrial cancers remains unknown. ChIP-qPCR experiments are ongoing to assess MAXH28R binding at the promoters of differentially expressed genes. Biologic validation of MAXH28R effects on differential expression of secreted proteins is being performed in the Ishikawa and RL95-2 endometrial cancer cell lines that stably express MAXH28R and MAXWT. The effects of reduced Laminin-β1 on integrin signaling and anchorage independent growth will be reported, as will effects of altered TGFβ expression on canonical and noncanonical TGFβ signaling. Citation Format: Craig M. Rush, Miranda L. Gardner, Caroline E. Sapp, Michael A. Freitas, Paul J. Goodfellow. Characterizing the MAXH28R secretome in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3335.

Regulation of matriptase and HAI-1 system, a novel therapeutic target in human endometrial cancer cells.

The effects of specific and non-specific regulation of matriptase on endometrial cancer cells in vitro were investigated. Messenger ribonucleic acid (mRNA) and protein expression of matriptase and hepatocyte growth factor activator inhibitor-1 (HAI-1) in RL-952, HEC-1A, and HEC-1B endometrial cancer cells were detected by real-time quantitative PCR (RT-qPCR) and western blot. The cells were infected with lentivirus-mediated small-interfering RNA (siRNA) targeted on matriptase (MA-siRNA) or treated with different cisplatin (DDP) concentrations. After treatment, invasion, migration, and cellular apoptosis were analyzed. Matriptase mRNA and protein expression significantly decreased to 80% after infection with MA-siRNA (P < 0.01), and scratch and trans-well chamber assays showed significant inhibition of invasiveness and metastasis. Upon incubation with cisplatin at concentrations higher than the therapeutic dose for 24 h, the expressions of matriptase and HAI-1 significantly decreased (P < 0.001). Moreover, the invasiveness, metastasis, and survival rate of HEC-1A and RL-952 endometrial cancer cells were significantly decreased (P < 0.001) due to the down-regulation of matriptase and HAI-1 upon increasing cisplatin concentration. However, a slight increase in matriptase and HAI-1 expression was observed in cells treated with low cisplatin concentration (P = 0.01). Moreover, matriptase expression was associated with metastasis and invasiveness. Down-regulation of matriptase by specific Ma-SiRNA or non-specific cisplatin in matriptase/HAI-1–positive endometrial cancer cells showed promising therapeutic features.

Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients.

BackgroundProstaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer.MethodsWe analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3.ResultsEP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells.ConclusionsEP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy.

The NMU signaling controls cancer progression of human RL95-2 endometrial cancer cells through cell motility maintenance

The NMU signaling controls cancer progression of human RL95-2 endometrial cancer cells through cell motility maintenance

Abstract 44: Enhancement endometrial cancer cells malignant transformation by mutual interaction with mesenchymal stem cells through TGF-beta and SDF-1 signaling pathways

Abstract Designated to cyclic shedding, endometrial glandular stroma is rich for mesenchymal stem cells and may play crucial roles in cancer invasion. This study aimed to explore the interaction between endometrial cancer cells and endometrial mesenchymal stem cells (HEMSC). HEMSC were derived from human endometrium and were co-cultured with RL95-2 endometrial cancer cells. Transformation phenotypes including proliferation, migration, anchorage independent growth, colony formation and invasion were characterized. Cancer associated transforming growth factor (TGF)-beta1 and mesenchymal cell associated stromal cell-derived factor (SDF) and their down-stream signaling pathways were investigated. HEMSC had a similar surface marker profile as bone marrow mesenchymal stem cells, i.e. CD44(+), CD90(+) and CD34(-) and CD45(-) and readily be inducible to osteogenic and adipogenic differentiations. Upon coculture with HEMSC, RL95-2 cell secreted TGF-beta 1 which subsequently induced SDF1 secretion from HEMSC. The mutual interaction of these factors is associated with enhancement of cancer cell migration through the CXCR4 receptor and downstream transducer and activator of Stat3, with resulting over-expression of alpha-SMA and cytoskeleton re-organization. The study observed unprecedented interactions between cancer cells and mesenchymal stem cells with resulting enhancement of malignant transformation through mutual activation of cytokines and downstream signals. Mesenchymal stem cells either in adjacent to the lesion or home to the lesion can therefore be important activators of carcinogenesis and good therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 44. doi:1538-7445.AM2012-44

DNMT Inhibitors and HDAC Inhibitors Regulate E-Cadherin and Bcl-2 Expression in Endometrial Carcinoma in vitro and in vivo

Background: The effect of histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) on proliferation of endometrial cancer (EC) cells in vitro and in vivo was investigated. Methods: Changes in methylation of the CDH1 promoter in HDACI- and DNMTI-treated HEC-1-B and RL-952 EC cells were detected. Nude mice with xenografted implants of human EC HEC-1-B cells were treated with valproic acid (VPA) and decitabine (DAC) and evaluated for tumor growth, CDH1 and Bcl-2 mRNA levels. Results: DAC, VPA and suberoylanilide hydroxamic acid (SAHA) inhibited proliferation, induced cell cycle arrest and enhanced the apoptotic index in both cell lines, DAC, VPA and SAHA upregulated E-cadherin mRNA and protein levels and downregulated Bcl-2 mRNA levels in vitro. DAC and VPA inhibited tumor growth, upregulated CDH1 mRNA and downregulated Bcl-2 mRNA levels in vivo. Conclusions: A combination of HDACIs and DNMTIs suppresses the growth of EC, which is likely mediated by upregulation of E-cadherin and downregulation of Bcl-2.

Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

Abstract RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo- p -dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC 50 s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.

7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system.

Acrp30 inhibits leptin-induced metastasis by downregulating the JAK/STAT3 pathway via AMPK activation in aggressive SPEC-2 endometrial cancer cells

Obesity is a well-established risk factor for endometrial cancer, due in part to the adipokines generated by adipose tissue, such as adiponectin (also known as Acrp30) and leptin, which are associated with many endocrine-related cancers. Recent reports suggested that Acrp30 inhibits leptin-stimulated cell proliferation in HEC-1A and RL95-2 endometrial cancer cell lines, and that the serum leptin/Acrp30 ratio plays an important role in endometrial cancer development. We explored whether Acrp30 could reverse the leptin-induced metastasis phenotype in the SPEC-2 endometrial cancer cell line. Transcripts for Acrp30 receptors (AdipoR1 and AdipoR2) and leptin receptor (Ob-Rb) were detected by quantitative real-time RT-PCR (qRT-PCR) in six endometrial cancer cell lines. Leptin (1µg/ml) treatment stimulated SPEC-2 cell proliferation by inducing cell cycle arrest and apoptosis, while Acrp30 (10µg/ml) treatment inhibited the growth of SPEC-2 cells. Importantly, Acrp30 was able to inhibit leptin-induced SPEC-2 cell proliferation. Leptin promoted SPEC-2 cell invasion in a Matrigel transwell assay, while Acrp30 partly suppressed the invasion stimulated by leptin. To investigate the molecular mechanism underlying this phenomenon, we monitored the AMPK and JAK/STAT3 signaling pathways by western blotting and cell immunofluorescence. Acrp30 reduced leptin-induced STAT3 phosphorylation and nuclear translocation via activation of the MAPK pathway. AG490 (JAK/STAT3 inhibitor) reduced MMP-2 and MMP-9 protein levels in cells treated with leptin, while IL-6 (JAK/STAT3 stimulator) and Compound C (AMPK inhibitor) elevated MMP-2 and MMP-9 protein levels in cells treated with Acrp30. In conclusion, we demonstrated that Acrp30 effectively reversed the invasion stimulated by leptin, and AMPK and JAK/STAT3 pathways mediated the invasive phenotype of SPEC-2 cells.

Aberrant expression and possible involvement of the leptin receptor in endometrial cancer

Objective. Leptin and its receptor are the key players in the regulation of energy balance and body weight control. However, their roles in gynecological malignancies are mostly unclear. In this study, we analyzed the expression and possible involvement of leptin and the leptin receptor in the pathogenesis of endometrial cancer. Methods. Radioimmunoassay was performed to analyze the serum leptin levels in the endometrial cancer patients, while RT-PCR, immunoblotting, and immunohistochemistry techniques were applied to study the expression of leptin receptor in the endometrioid-type endometrial cancer tissues. Furthermore, BrdU labeling followed by immunofluorescent analysis was used to analyze the effect of leptin receptor overexpression on endometrial cancer cell proliferation. Results. Serum leptin levels are elevated in endometrial cancer patients, but show no significant difference to those of normal controls when normalized by body mass index. On the other hand, lower expression levels of leptin receptor short form (Ob-Ra) were observed in most endometrial cancer tissues, especially in the poorly differentiated ones, and the forced expression of Ob-Ra in RL95-2 endometrial cancer cells prevented them from entering the S-phase. Conclusion. In summary, our data demonstrates for the first time that the leptin receptor is aberrantly expressed in endometrial cancer tissues and is possibly involved in the pathogenesis of endometrial cancer.

Characterization of cytosolic estrogen sulfotransferase from RL95-2 endometrial cancer cells.

Estrogen sulfoconjugation previously was reported in normal endometrium and in RL95-2 cells, a cell line derived from a human endometrial cancer maintained in continuous in vitro culture. In the present study the estrogen sulfurylation activity in the cytosolic fraction of RL95-2 cells was characterized using [3H]estrone as substrate. Estrone sulfate was separated from unreacted estrone by thin layer chromatography. Activity was proportional to cytosol concentration, with a pH optimum at pH 8. There was marked temperature dependence between 24 and 40 C. The apparent Km for estrone conjugation was 3.6 nM, with a maximum velocity of 135 fmol/micrograms DNA . h. No complex kinetic behavior was found at estrone concentrations up to 1 microM. The apparent Km for the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate was 0.6 microM. Inhibition experiments demonstrated that the sulfurylating activity studied under these conditions was specific for estrogens. Only estradiol and estriol, in addition to estrone itself, inhibited conjugation to any significant degree. Dehydroepiandrosterone had only 1% the inhibitory activity of estrone. Other androgens, corticoids, progestins, phenols, nonsteroidal estrogens and antiestrogens, and bile acids had no significant effects on the sulfurylation of estrone. An estrogen-sulfoconjugating activity with the characteristics of estrogen sulfotransferase (EST) was demonstrated in RL95-2 cells. The Km of EST for estrone in the RL95-2 cells closely approximated the value reported for the enzyme in normal endometrium. The affinity of EST for estrogens is within the range of the Kd of estrogen receptor and of the physiological concentrations of estrogens reported in the endometrium, suggesting that EST could serve as a regulator of intracellular estrogen levels.