KLE Cells Research Articles

B3GNT3 acts as a carcinogenic factor in endometrial cancer via facilitating cell growth, invasion and migration through regulating RhoA/RAC1 pathway-associated markers.

Aberrant expression of beta-1,3-N-acetylglucosaminyltransferase-3 (B3GNT3) has been frequently clarified in various cancers, however, its role in endometrial cancer (EC) has not been assessed in detail. This study aimed to investigate the biological role of B3GNT3 in EC and simply explored the detailed mechanism. The EC RNA-Seq dataset from TCGA database was applied to evaluate the expression of B3GNT3 and assess its role on prognostic value. HEC-1-A and KLE cell lines of EC were used to perform loss- and gain-of-function B3GNT3 assays respectively. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure the mRNA and protein levels of indicated molecules respectively. Cell counting kit-8, clone formation tests, and Transwell assay served to determine the changes of proliferative, invasive and migratory abilities of EC cells after altering the expression of B3GNT3. B3GNT3 was found to be highly expressed in EC tissues compared to normal tissues according to the online public databases, which confirmed by the following qRT-PCR in 3 EC cell lines. Besides, high B3GNT3 expression presented a worse overall survival in EC patients as compared with low B3GNT3 expression group. Furthermore, functional experiments in vitro indicated that B3GNT3 could facilitate the cell growth, invasion and migration. Moreover, we found that downregulation of B3GNT3 significantly reduced the expression level of GTP-RhoA and GTP-RAC1, whereas upregulation of B3GNT3 presented the opposite results. The results of current study demonstrate that B3GNT3 acts as an oncogene that promotes EC cells growth, invasion and migration possibly through regulating the RhoA/RAC1 signaling pathway-related markers, suggesting that B3GNT3 may be a candidate biomarker for EC therapeutic intervention.

Mechanisms of Cisplatin in Combination with Repurposed Drugs against Human Endometrial Carcinoma Cells.

Although endometrial carcinoma is one of the most common gynecological malignancies worldwide, its precise etiology remains unknown. Moreover, no novel adjuvant and/or targeted therapies are currently being developed to achieve greater efficacy for endometrial cancer patients who develop chemotherapeutic drug resistance. In this study, we used three human endometrial cancer cell lines, RL95-2, HEC-1-A, and KLE, to investigate the responsiveness of cisplatin alone and in combination with potential repurposed drugs. We first found that RL95-2 cells were more sensitive to cisplatin than HEC-1-A or KLE cells. The cytotoxicity of cisplatin in RL95-2 cells may reflect its ability to perturb the cell cycle, reactive oxygen species production and autophagy as well as to induce senescence and DNA damage. Similar effects, although not DNA damage, were also observed in HEC-1-A and KLE cells. In addition, downregulation of p53 and/or cyclin D1 may also impact the responsiveness of HEC-1-A and KLE cells to cisplatin. We also observed that resveratrol, trichostatin A (TSA), caffeine, or digoxin increased the apoptotic process of cisplatin toward RL95-2 cells, while amiodarone or TSA increased its apoptotic process toward HEC-1-A cells. The combination index supported the assertion that the combination of cisplatin with caffeine, amiodarone, resveratrol, metformin, digoxin, or TSA increases the cytotoxicity of cisplatin in HEC-1-A cells. These findings suggest potential strategies for enhancing the efficacy of cisplatin to overcome drug resistance in endometrial carcinoma patients.

Sirtuin2 correlates with lymph node metastasis, increased FIGO stage, worse overall survival, and reduced chemosensitivity to cisplatin and paclitaxel in endometrial cancer.

This study aimed to investigate the correlation of sirtuin2 (SIRT2) with clinical characteristics, prognosis in endometrial cancer (EC) patients, and its effect on chemosensitivity in EC cell lines. A total of 137 EC patients who underwent surgical resection were retrospectively enrolled. SIRT2 expression in tumor tissues (n = 137) and adjacent tissues (n = 61) was detected by immunohistochemistry (IHC) staining and evaluated by a semiquantitative scoring method. EC patients' clinical characteristics and survival data were collected. Besides, SIRT2 was modulated by plasmid transfection in EC cells, then their chemosensitivity to cisplatin and paclitaxel was evaluated. SIRT2 was increased in tumor tissues compared with adjacent tissues (reflected by both IHC score and high-expression ratio, both P < 0.001). Meanwhile, tumor SIRT2 was positively correlated with lymph node metastasis (P = 0.037) and the International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.044), but not other clinical characteristics. Moreover, tumor SIRT2 high expression was correlated with worse overall survival (OS) (P = 0.023), while it could not independently predict OS (P = 0.090, hazard ratio = 2.782). Besides, both mRNA and protein levels of SIRT2 were increased in Ishikawa (P = 0.035) and KLE (P < 0.001) cells compared with human endometrial epithelial cells. SIRT2 overexpression decreased chemosensitivity to cisplatin and paclitaxel in Ishikawa cells, while SIRT2 knockdown increased chemosensitivity to cisplatin and paclitaxel in KLE cells (all P < 0.05). SIRT2 correlates with lymph node metastasis, increased FIGO stage, worse OS, and reduced chemosensitivity to cisplatin and paclitaxel in EC.

Silencing of PTPN18 Induced Ferroptosis in Endometrial Cancer Cells Through p-P38-Mediated GPX4/xCT Down-Regulation.

BackgroundEndometrial cancer (EC) is the fourth most common neoplasm and the eighth leading cause of cancer death in females worldwide. PTPN18 is a member of the protein tyrosine phosphatases (PTP) family, which is associated with the occurrence and progression of various human cancers. PTPN18 was up-regulated in endometrial cancer tissues and high level of PTPN18 promoted proliferation and metastasis of EC cells.MethodsThe expression of PTPN18, GPX4 and xCT in endometrial cancer tissues and KLE cells was detected by immunohistochemistry and Western blot, respectively. Lentiviral transfection were used to silence PTPN18 level in KLE cells. The Ros level in KLE cells was examined by ELISA assay.ResultsIn the present study, we found that silencing of PTPN18 induced ferroptosis in KLE endometrial cancer cells. PTPN18 knockdown increased intracellular ROS level and down-regulated GPX4 and xCT expression. Besides, silencing of PTPN18 also induced the expression of p-p38.ConclusionWe concluded that silencing of PTPN18 might induce ferroptosis by targeting the p-p38/GPX4/xCT axis. The results provide critical insight into the application of PTPN18 knockdown in EC intervention.

Abstract PO042: ROR2 is epigenetically regulated in endometrial cancer

Abstract BACKGROUND: The Wnt signalling receptor ROR2 has been identified as a potential target in numerous cancers, however its exact role remains controversial. ROR2 has been shown to play distinct roles in regards to tumorigenesis in different tumour types. Our previous study suggested a potential tumour suppressor role for ROR2 in endometrial cancer (EC), however the mechanism associated with ROR2 repression in EC remains unclear. ROR2 has been found to be epigenetically inactivated in colorectal cancer. OBJECTIVE: The aim of this study was to investigate the role of ROR2 in EC, and to determine if ROR2 expression in EC was regulated epigenetically. METHODS: The correlation between ROR2 mRNA expression levels and clinicopathological parameters, including overall survival (OS) was analysed in The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA-UCEC, n=546) cohort and GEO datasets (GSE17025, n=91). Immunohistochemistry (IHC) of ROR2 was performed on a large EC patient cohort (Australia-wide population-based Australian EC Study, ANECS, n=499). The correlation between ROR2 staining intensity (0-3) and clinicopathological parameters as well as 5-year progression free survival (PFS) and OS was analysed. Cox regression was also applied to analyse the impact of selected covariates (age, BMI, FIGO stage and grade) on the PFS and OS. Four EC cell lines (KLE, MFE-296, Ishikawa and ARK-1) and eight clinical EC samples were analysed for ROR2 methylation via COmbined Bisulphite Restriction Analysis (COBRA) and bisulphite genomic sequencing (BGS). RESULTS: ROR2 promoter methylation or reduced ROR2 expression were both found to correlate with lower OS, high grade and serous EC in the TCGA-UCEC and GEO dataset at p&amp;lt;0.05 level. In our ANECS cohort, the same trend of correlation was observed of ROR2 expression and grade (p=0.062). No significant correlation was observed for ROR2 expression with OS or PFS in this cohort at univariate or multivariable survival analysis. ROR2 was epigenetically silenced by promoter methylation in KLE and ARK-1 cell lines. There was also a significant correlation between ROR2 expression level and promoter methylation in our patient cohort (r=-0.79, p=0.02). CONCLUSION: ROR2 plays a tumour suppressor role in EC and is epigenetically suppressed with the development of disease. It may therefore be a diagnostic or therapeutic candidate for EC. Citation Format: Dongli Liu, Luis Enriquez, Benjamin Daniels, Tracy O'Mara, Katrina Tang, Caroline Ford. ROR2 is epigenetically regulated in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO042.

LINC00665 enhances tumorigenicity of endometrial carcinoma by interacting with high mobility group AT-hook 1

BackgroundEndometrial carcinoma is a frequently diagnosed cancer among females. LncRNAs are reported to be associated with various cancers. Their biological roles in endometrial carcinoma progression is an emerging scientific area. LINC00665 can exert a significant role in many cancers. However, its potential function in endometrial carcinoma is still poorly known.MethodqRT-PCR was carried out to test expression of LINC00665 and HMGA1. Western blot analysis was carried out to detect protein expression of HMGA1. Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8) and EdU assay. Flow cytometry assay was used to determine cell apoptosis and cell cycle. Wound healing and transwell invasion assay was carried out to test cell migration and invasion. Immunohistochemical staining and HE staining were conducted to assess Ki-67 and tumor growth respectively.ResultsExpression of LINC00665 in clinical endometrial carcinoma tissues and cells was obviously up-regulated. Loss of LINC00665 could repress endometrial carcinoma cell viability, induce cell apoptosis and block cell cycle in G1 phase. KLE and HHUA cell migration and invasion ability were depressed by LINC00665 shRNA. Decrease of LINC00665 suppressed endometrial carcinoma tumorigenicity in vivo. RIP assay proved that LINC00665 directly bound with HMGA1 protein. shRNA of HMGA1 obviously restrained endometrial carcinoma cell growth and cell invasion.ConclusionsLINC00665 might promote endometrial carcinoma progression by positively modulating HMGA1.

Biogenesis of copper nanoparticles (Cu-NPs) using leaf extract of Allium noeanum, antioxidant and in-vitro cytotoxicity

In this research, we formulated new chemotherapeutic copper nanoparticles (Cu NPs) containing Allium noeanum Reut. ex Regel leaf for treating human endometrial cancer. For investigating the antioxidant activitiy, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used. MTT test was used on normal (Human umbilical vein endothelial cells (HUVECs)) and human endometrial cancer (Ishikawa, HEC-1-A, HEC-1-B, and KLE) cell lines for comparing the anti-human endometrial cancer properties of Cu(NO3)2, A. noeanum leaf aqueous extract, and copper nanoparticles. Copper nanoparticles had high cell death and anti-human endometrial cancer effects against Ishikawa, HEC-1-A, HEC-1-B, and KLE cell lines. The IC50 of A. noeanum leaf aqueous extract and copper nanoparticles against HEC-1-B cell line were 548 and 331 µg/mL, respectively; against HEC-1-A cell line were 583 and 356 µg/mL, respectively; against KLE cell line were 609 and 411 µg/mL, respectively; and against Ishikawa cell line were 560 and 357 µg/mL, respectively. Among the above cell lines, the best result of anti-human endometrial cancer properties of copper nanoparticles was gained in the cell line of HEC-1-B. This study indicated excellent anti-human endometrial cancer potentials of copper nanoparticles containing A. noeanum in the in vitro condition.

Medroxyprogesterone Reverses Tolerable Dose Metformin-Induced Inhibition of Invasion via Matrix Metallopeptidase-9 and Transforming Growth Factor-β1 in KLE Endometrial Cancer Cells.

This study was performed to evaluate the anticancer effects of tolerable doses of metformin with or without medroxyprogesterone (MPA) in endometrial cancer cells. Cell viability, cell invasion, and levels of matrix metallopeptidase (MMP) and transforming growth factor (TGF)-β1 were analyzed using three human endometrial adenocarcinoma cell lines (Ishikawa, KLE, and uterine serous papillary cancer (USPC)) after treatment with different dose combinations of MPA and metformin. Combining metformin (0, 100, 1000 µM) and 10 µM MPA induced significantly decreased cell viability in a time- and dose-dependent manner in Ishikawa cells, but not in KLE and USPC cells. In KLE cells, metformin treatment alone significantly inhibited cell invasion in a dose-dependent manner. The inhibitory effect of metformin was reversed when 10 µM MPA was combined, which was significantly inhibited again after treatment of MMP-2/9 inhibitor and/or TGF-β inhibitor. Changes of MMP-9 and TGF-β1 according to combinations of MPA and metformin were similar to those of invasion in KLE cells. In conclusion, the anticancer effects of tolerable doses of metformin varied according to cell type and combinations with MPA. Anti-invasive effect of metformin in KLE cells was completely reversed by the addition of MPA; this might be associated with MMP-9 and TGF-β1.

MDM4 is targeted by miR-449b-5p to promote the proliferation of endometrial carcinoma.

Previous studies have shown the involvement of microRNA-449b-5p (miR-449b-5p) and MDM4 in tumor development. This study aims to illustrate the role of miR-449b-5p in inhibiting proliferative capacity of endometrial carcinoma (EC) by targeting MDM4. Expression levels of miR-449b-5p and MDM4 in tumor tissues and paracancerous ones of EC patients were determined. Relationships between their levels and clinical parameters of EC patients were analyzed. Subsequently, regulatory effects of miR-449b-5p and MDM4 on proliferative capacities in KLE and HEC-1B cells were assessed by cell counting kit-8 (CCK-8) and colony formation assay, respectively. Thereafter, in vivo xenograft models were established in nude mice administrated with KLE cells overexpressing MDM4 or those with miR-449b-5p knockdown. Then, tumor weight and tumor volume were measured after mouse sacrifice. Finally, the interaction between miR-449b-5p and MDM4 was explored by Luciferase assay. It was found that MDM4 was upregulated and miR-449b-5p was downregulated in EC tissues. Highly expressed MDM4 and lowly expressed miR-449b-5p were unfavorable to prognosis in EC patients, manifesting as a larger tumor size, more advanced tumor stage and lower overall survival. Besides, overexpression of MDM4 enhanced in vitro proliferative capacity in EC cells and in vivo tumorigenesis in nude mice bearing EC. Similarly, knockdown of miR-449b-5p yielded similar results. Luciferase assay confirmed that MDM4 was the target gene binding to miR-449b-5p, and its level was negatively correlated with miR-449b-5p level in EC. MiR-449b-5p and MDM4 are downregulated and upregulated in EC species, respectively. They are closely linked to tumor size, tumor stage and overall survival in EC patients. Through negatively regulating MDM4 level, miR-449b-5p inhibits proliferative capacity in EC cells.

Abstract 2942: ROR1: A novel target in endometrial cancer

Abstract Background Endometrial cancer is one of the fastest rising cancers in women, and despite overall high survival, new therapies are required for women with aggressive subtypes of the disease. ROR1 and ROR2 are Wnt receptor tyrosine kinases whose expression is altered in a range of cancers. This study aimed to investigate the role of ROR1/2 in endometrial cancer (EC). Method Immunohistochemistry (IHC) of ROR1 and ROR2 was performed on an EC patient cohort (Australia-wide population-based Australian EC Study, ANECS, n=499). The association between ROR1/2 staining intensity (0-3) and clinicopathological parameters was analyzed, as well as association with 5-year progression free survival (PFS) and overall survival (OS). Cox multivariate regression was also applied to analyze the impact of selected covariates (age, BMI, FIGO stage, grade and subtypes) on the PFS and OS. The functional effect of ROR1 and ROR2 modulation was further investigated in two EC cell lines–KLE (high ROR1, low ROR2) and MFE-296 (low ROR1, high ROR2). For KLE, ROR1 silencing and ROR2 overexpression were performed. In contrast, ROR2 silencing and ROR1 overexpression were performed in MFE-296. ROR1 or ROR2 silencing was achieved via transfection with ROR1 or ROR2 siRNA. ROR1 pCMV3 plasmid or ROR2 pFLAG plasmid were transfected for ROR1 or ROR2 overexpression. All the conditions were compared to the negative control which was prepared by transfecting both non-targeting siRNA and pCMV3-NH plasmid. Effects on proliferation, adhesion, migration and invasion were measured. Result In the patient cohort, ROR1 expression level was significantly associated with tumor grade (p=0.004) and moderately associated with FIGO stage (p=0.057). A significant decrease in OS and PFS was observed in patients with high ROR1 expression (p=0.045 and 0.003 respectively). This correlation was not lost when filtering against multiple parameters including age, BMI, FIGO stage and tumor grade in COX regression (p=0.049). No significant correlation was observed for ROR2 expression with OS or PFS, though high ROR2 showed a trend towards better PFS. ROR1 downregulation or ROR2 upregulation in KLE cells decreased cell proliferation after 72 hours. The combination of the two further reduced cell proliferation significantly after 48h and 72h (p=0.04 and 0.004 respectively). Proliferation was not significantly altered in MFE-296, although ROR1 overexpression showed a trend for increasing cell proliferation after 72h. ROR1 silencing in KLE decreased the migration ability moderately (p=0.059) and its combination with ROR2 overexpression further reduced migration significantly (p=0.038). No significant change in invasion or adhesion was observed. Conclusion This study confirms the oncogenic role ROR1 plays in EC, which warrants the future application of ROR1-targeting therapies in EC patients. The role of ROR2 is more complex in EC and may be context dependent. Citation Format: Dongli Liu, Kate Gunther, Benjamin Daniels, Tracy O'Mara, Katrina Tang, Australian National Endometrial Cancer Study Group, Caroline Ford. ROR1: A novel target in endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2942.

Actin‑like protein 8 executes a promoting function in the malignant progression of endometrial cancer: identification of a promising biomarker.

Endometrial cancer (EC) is generally considered as a disease that affects older women. We attempt to explore the role of actin‑like protein 8 (ACTL8) in EC and how it achieves its function. Based on the data from The Cancer Genome Atlas (TCGA), we found that ACTL8 expression was up-regulated in EC tissues and correlated with shorter overall survival of EC patients. ACTL8 expression was significantly associated with age, clinical-stage, or grade. Cox proportional hazards model analysis revealed that ACTL8 expression, grade, and clinical-stage were promising independent prognostic factors of EC. Knockdown of ACTL8 repressed the proliferative, migrating and invading capabilities of human EC cell lines KLE and Ishikawa. Silencing ACTL8 up-regulated the negative cell cycle regulator p21 and epithelial marker E-cadherin, and down-regulated the positive cell cycle regulator Cyclin A, mesenchymal markers MMP-9 and N-cadherin in KLE cells. Collectively, these outcomes illustrated that ACTL8 might act as a tumor facilitator during EC progression.

Effect of shRNA-mediated regulation of S100A4 gene expression on proliferation and apoptosis of KLE endometrial cancer cells.

To investigate the effect of shRNA-regulated S100A4 expression on the proliferation and apoptosis in KLE endometrial cancer cells. S100A4-OVER and S100A4-shRNA were transfected into KLE endometrial cancer cells using lentiviral sh-RNA technology. Passive OVER-NC cell line and shRNA-NC cell line were used as a negative control group and non-transfected Control cell line as a blank control group. After 48h of transfection, the expressions of S100A4 and protein were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. CCK-8 detection and flow cytometer were used to detect cell proliferation and apoptosis, respectively. Compared with the normal control group and the negative control group, the transfection efficiency and shRNA targeting of the shRNA-interfered S100A4 gene were verified at the levels of mRNA and protein expression. The expression of the disrupted S100A4 gene at S100A4 mRNA and protein levels in endometrial cancer cells was determined. The proliferation efficiency of KLE cells in S100A4-OVER group was significantly higher than that in other four groups; the proliferation rate of S100A4-shRNA cells decreased slightly;, the apoptotic rate of KLE cells in S100A4-shRNA group increased significantly, and the apoptotic rate of KLE cells in S100A4-OVER group decreased compared with NC group. Specific regulation of S100A4 gene expression:, the enhanced expression of the S100A4 gene may promote the proliferation of KLE endometrial cancer cells; the inhibited expression of the S100A4 gene may promote the apoptosis of KLE endometrial cancer cells. S100A4 expression is closely related to the biological characteristics of endometrial cancer.

Repurposing of antipsychotics perphenazine for the treatment of endometrial cancer

Endometrial cancer (EC) is one of the most common and fatal gynecological cancers worldwide, but there is no effective treatment for the EC patients of progesterone resistance. Repurposing of existing drugs is a good strategy to discover new candidate drugs. In this text, perphenazine (PPZ), approved for psychosis therapy, was identified as a potential agent for the treatment of both progesterone sensitive and resistant endometrial cancer for the first time. Specifically, perphenazine exhibited good cell proliferation inhibition in Ishikawa (ISK) and KLE cell lines according to the CCK-8 assay and colony formation assay. It also reduced the cell migration of ISK and KLE cell lines in the light of the transwell migration assay. Annexin-V/PI double staining assay suggested that perphenazine could effectively induce ISK and KLE cell apoptosis. Moreover, results of western blot assay indicated perphenazine obviously inhibited the phosphorylation of Akt. Delightedly, PPZ also could significantly attenuate xenograft tumor growth at both 3 mg/kg and 15 mg/kg in mice without influencing the body weights.

Knockdown of Trop2 inhibits proliferation and migration and induces apoptosis of endometrial cancer cells via AKT/β-catenin pathway.

Endometrial cancer (EC) is the most common gynaecologic malignancy in western countries and has been reported to account for about 7% of female malignant tumours and 20% to 30% of female genital system malignant tumours. Accumulating evidence showed the expression of human trophoblast cell surface antigen 2 (Trop2) was abnormal in many cancers; however, the expression and role of Trop2 in EC are not clear. The Trop-2 protein expression was detected by western blot in EC cells. Cell proliferation, apoptosis, and migration were measured by CCK-8, flow cytometry, and Transwell assay, respectively. The epithelial mesenchymal transition (EMT) and AKT/β-catenin signalling pathway-related proteins in EC cell lines were detected by western blot assay following Trop2 gene silencing. The present study revealed that the Trop2 protein was highly expressed in EC cell lines compared with human endometrial epithelial cells. The Trop2 mRNA and protein were obviously decreased following transfection with Trop2-siRNA sequence in KLE and Ishikawa cells. Meanwhile, Trop2 gene silencing in KLE and Ishikawa cells strongly inhibited cell proliferation and migration and increased cell apoptosis. Investigation into the molecular mechanism indicated that the Trop2 gene silencing suppressed EMT and AKT/β-catenin signalling pathway activation. SIGNIFICANCE OF THE STUDY: These findings suggested that Trop2 silencing inhibited EC cell proliferation and migration and promoted cell apoptosis. The mechanism might be related to the inhibition of the AKT/β-catenin signalling pathway in EC cells. Therefore, Trop2 may be a potential therapeutic target for the treatment of EC.

RETRACTED: LncRNA PCAT1 promotes metastasis of endometrial carcinoma through epigenetical downregulation of E-cadherin associated with methyltransferase EZH2

More than 140 thousands of women suffer from endometrial carcinoma in the worldwide, and over 40 thousand of the patients die before and after in surgery and chemoradiotherapy because of its metastasis. However, its molecular mechanism is much less known compared to other cancers. In this study, we demonstrated that long non-coding RNA PCAT1 is dramatically increased in the tissues and plasma from endometrial carcinoma (EC) (n = 100, all p < 0.001) controlled by its paracancerous tissue, and cell lines including RL-952, HEC-1-B, KLE, Ishikawa, and AN3CA compared to the cells from normal endometrium (all p < 0.001). When lncRNA PCAT1 was knocked-down, the KLE and AN3CA cells exhibited slow capability on proliferation and colony formation in vitro. With the silence of lncRNA PCAT1, the cells were markedly inhibited on migration and invasion in vitro (all p < 0.001), which were confirmed on the EC patient subjects. When expressions of lncRNA PCAT1 were interfered in the cells, expressions of E-cadherin but not N-cadherin and Vimentin were significantly promoted with a strong up-regulation accompanied by nearly completed recoveries on migration and invasion (all p < 0.001). In order to analyze the association of lncRNA PCAT1 and E-cadherin, we silenced the expressions of both genes and unveiled that EC migration and invasion were significantly congested (all p < 0.001). Importantly, we found that the E-cadherin down-regulation caused by lncRNA PCAT1 associates with histone methyltransferase EZH2. When over-expression of EZH2 was applied in the PCAT1 silenced cells, the expression of E-cadherin experienced significant decrease in the cell lines. Reversely, when expression of EZH2 was annulled in the PCAT1 silenced cells, the expression of E-cadherin was significantly boosted in the cells (all p < 0.001). Furthermore, the interaction of lncRNA PCAT1 and EZH2 were approved with immunoprecipitation. Our data demonstrated that the methyltransferase EZH2 related up-regulation of lncRNA PCAT1 along with down-regulation of E-cadherin could be essential in oncogenesis of endometrial carcinoma in both EC cells and patient subjects. These compact data suggest that combination of lncRNA PCAT1, EZH2 and E-cadherin could provide valued information for efficient EC diagnostics, which would propose a potential target for EC treatment with EZH2i on methyltransferation.

Osthole inhibited cell proliferation and induced cell apoptosis through decreasing CPEB2 expression via up-regulating miR-424 in endometrial carcinoma

Objective: Endometrial carcinoma (EC) was the fourth female malignancies in developed countries. Given that the prognosis of EC is extremely poor, it is vital to investigate its pathogenesis and effective therapeutic targets. However, the mechanism of osthole in EC remains unknown.Materials and methods: Firstly, the different doses of osthole (0, 50, 100, and 200 μM) were used to treat the Ishikawa and KLE cells. The cell proliferation, apoptosis, and cell cycle were measured by cell counting kit-8 (CCK-8), Annexin V-FITC/PI, and cell cycle assays. The apoptosis-related protein levels were examined by western blot. The miR-424 levels in Ishikawa and KLE cells were assessed by quantitative RT-PCR (qRT-PCR). Also, the binding of miR-424 and cytoplasmic polyadenylation element binding protein 2 (CEPB2) was detected by the luciferase reporter assay.Results: In this study, the increasing dose of osthole inhibited proliferation and induced apoptosis of Ishikawa and KLE cells. Moreover, the increasing dose of osthole up-regulated miR-424 and down-regulated the expression of CPEB2. CPEB2 was proved to be the target gene of miR-424. Interestingly, the over-expression of CPEB2 could reverse the changes of osthole-induced proliferation and apoptosis of Ishikawa and KLE cells.Conclusions: In summary, we provided first evidences that osthole inhibited proliferation and induced apoptosis through up-regulating miR-424 to inhibit expression of CPEB2 in EC. Our findings indicated that osthole might act as a novel and potential therapeutic agent for the treatment of EC.

CARLo-5 as an oncogenic gene in endometrial carcinoma.

The long noncoding RNA CARLo-5 is dysregulated in multiple types of human cancers. High CARLo-5 is a promising predictive factor for various cancers, including endometrial carcinoma (EC). Our previous study showed that the expression level of CARLo-5 was associated with advanced FIGO stage (The International Federation of Gynecology and Obstetrics), lymph node metastasis, and the poor survival of patients with EC. In the present study, we demonstrated that the downregulation of CARLo-5 could affect the proliferation, cell cycle, migration, and invasion of EC cell lines HEC-1B and KLE cells. The oncogenic activity of CARLo-5 was also confirmed with in vivo data. Mechanistically, CARLo-5 could affect the expression of CDK/CDKN1A and MMP2/9, which have been reported to be regulated by CARLo-5 and associated with cell cycle and motility. In conclusion, this study is the first to discover the biological function and mechanism of CARLo-5 in regulating the biological characteristics of EC cells. Targeting CARLo-5 and its pathway might provide new biomarkers or potential therapies target for patients with EC. Environ. Mol. Mutagen. 61:256-265, 2020. © 2019 Wiley Periodicals, Inc.

Medroxyprogesterone acetate causes the alterations of endoplasmic reticulum related mRNAs and lncRNAs in endometrial cancer cells

BackgroundProgestin is effective to promote endometrial cancer (EC) cells apoptosis, however, continuous progestin administration causes low level of progestin receptor B (PRB), further resulting in progestin resistance. Here, we performed microarray analysis on Ishikawa cells (PRB+) treated with medroxyprogesterone acetate (MPA) to explore the molecular mechanism underlying the inhibitory influence of MPA on PRB+ EC cells.MethodsMicroarray analysis was performed by using Ishikawa cells (PRB+) treated with MPA. Differentially expressed mRNA and long noncoding RNAs (lncRNAs) were identified. Furthermore, the functions of these mRNAs and lncRNAs were predicted by functional enrichment analysis. QRT-PCR was further performed to verify the microarray data.ResultsA total of 358 differentially expressed genes and 292 lncRNAs were identified in Ishikawa cells (PRB+) treated with MPA. QRT-PCR verified these data. Functional enrichment analysis identified endoplasmic reticulum (ER) stress as the key pathway involved in the inhibitory effect of MPA on EC cells. And the ER stress apoptotic molecule CHOP and ER stress related molecule HERPUD1 were both highly expressed in Ishikawa cells (PRB+) treated with MPA. Co-expression analysis showed lnc-CETP-3 was highly correlated with CHOP and HERPUD1, suggesting it might participate in ER stress pathway-related EC cell apoptosis caused by MPA. In addition, compared with untreated cells, lnc-CETP-3, CHOP and HERPUD1 were significantly up-regulated in Ishikawa cells (PRB+) treated with MPA, whereas they have no statistical significance in KLE cells (PRB-).ConclusionsMPA may activate ER stress by progesterone-PRB pathway to up-regulate CHOP expression, which may be one of the molecular mechanisms underlying the inhibitory effect of MPA on EC cells with PRB+. Lnc-CETP-3 might be involved in this process. These findings may provide therapeutic targets for EC patients with PRB-, and resistance-related targets to increase the sensitivity of MPA on EC cells.

Novel dihydroartemisinin dimer containing nitrogen atoms inhibits growth of endometrial cancer cells and may correlate with increasing intracellular peroxynitrite

In the present study, a novel dimer, SM1044, selected from a series of dihydroartemisinin (DHA) derivatives containing nitrogen atoms comprising simple aliphatic amine linkers, showed strong growth inhibition in six types of human endometrial cancer (EC) cells, with half maximal inhibitory concentration (IC50) and 95% confidence interval (CI) < 3.6 (1.16~11.23) μM. SM1044 evoked apoptosis and activated caspase-3, −8 and −9 in a concentration- and time-dependent manner, and these effects were manifested early in RL95-2 compared to KLE cells, possibly correlated with the induction of intracellular ONOO−. Catalase and uric acid attenuated the growth inhibitory effects of SM1044 on EC cells, but sodium pyruvate did not. In vivo, the average xenograft tumour growth inhibition rates ranged from 35.8% to 49.9%, respectively, after 2.5 and 5.0 mg/kg SM1044 intraperitoneal treatment, and no obvious behavioural and histopathological abnormalities were observed in SM1044-treated mice in this context. SM1044 predominantly accumulated in the uteri of mice after a single injection. SM1044 displayed efficacy as a tumour suppressor with distinct mechanism of action and unique tissue distribution, properties that distinguish it from other artemisinin analogues. Our findings provide a new clue for artemisinin analogue against cancer.

Metformin Enhances Nomegestrol Acetate Suppressing Growth of Endometrial Cancer Cells and May Correlate to Downregulating mTOR Activity In Vitro and In Vivo.

This study investigated the effect of a novel progestin and its combination with metformin on the growth of endometrial cancer (EC) cells. Inhibitory effects of four progestins, including nomegestrol acetate (NOMAC), medroxyprogesterone acetate, levonorgestrel, and cyproterone acetate, were evaluated in RL95-2, HEC-1A, and KLE cells using cell counting kit-8 assay. Flow cytometry was performed to detect cell cycle and apoptosis. The activity of Akt (protein kinase B), mTOR (mammalian target of rapamycin) and its downstream substrates 4EBP1 (4E-binding protein 1) and eIF4G (Eukaryotic translation initiation factor 4G) were assayed by Western blotting. Nude mice were used to assess antitumor effects in vivo. NOMAC inhibited the growth of RL95-2 and HEC-1A cells, accompanied by arresting the cell cycle at G0/G1 phase, inducing apoptosis, and markedly down-regulating the level of phosphorylated mTOR/4EBP1/eIF4G in both cell lines (p < 0.05). Metformin significantly increased the inhibitory effect of and apoptosis induced by NOMAC and strengthened the depressive effect of NOMAC on activity of mTOR and its downstream substrates, compared to their treatment alone (p < 0.05). In xenograft tumor tissues, metformin (100 mg/kg) enhanced the suppressive effect of NOMAC (100 mg/kg) on mTOR signaling and increased the average concentration of NOMAC by nearly 1.6 times compared to NOMAC treatment alone. Taken together, NOMAC suppressing the growth of EC cells likely correlates to down-regulating the activity of the mTOR pathway and metformin could strengthen this effect. Our findings open a new window for the selection of progestins in hormone therapy of EC.