HHUA Cells Research Articles

A new labdane-type diterpenoid from leaves of Vitex rotundifolia

Vitex rotundifolia is a folk medicinal plant that has been used to treat various diseases. We have previously found that the extract of its leaves shows cytotoxic and epithelial mesenchymal transition inhibitory effect in HHUA endometrial cells. In this study, a new labdane-type diterpenoid 1 was isolated and its structure was elucidated by NMR and CD spectroscopic analysis followed by density functional theory calculations. Compound 1 showed cytotoxicity toward HHUA cells with an IC50 value of 6.4 μM, but did not inhibit phorbol ester-induced epithelial mesenchymal transition nor lipopolysaccharide-stimulated nitric oxide production.

Cell Morphology-Based Screening Identified Vitetrifolin D from <i>Vitex Rotundifolia</i> as an Inhibitor of Phorbol Ester–Induced Downregulation of E-Cadherin in HHUA Endometrial Cells

The epithelial–mesenchymal transition (EMT) of endometrial cells contributes to the development of endometrial cancer, endometriosis, and adenomyosis. We have recently reported that 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, induces EMT in HHUA endometrial cells cultured on collagen type I gels. HHUA cells showed an obvious morphological change from spheroids to scattered spindle cells during the TPA-induced EMT. In this study, we searched for inhibitors of the TPA-induced morphological change, and isolated vitetrifolin D from Vitex rotundifolia leaves. Vitetrifolin D suppressed the TPA-induced downregulation of E-cadherin, a major component of the epithelial adherens junction. Since vitetrifolin D showed a modest cytotoxicity at the concentration required to inhibit E-cadherin downregulation, it would not be suitable as a drug to treat endometrial disorders. However, our cell morphology-based screening could contribute to the discovery of new compounds that inhibit the loss of epithelial cell junctions.

12-O-Tetradecanoylphorbol 13-acetate promotes proliferation and epithelial-mesenchymal transition in HHUA cells cultured on collagen type I gel: A feasible model to find new therapies for endometrial diseases.

HHUA endometrial adenocarcinoma cells aggregated into spheroids when cultured on collagen type I gels. 12-O-Tetradecanoylphorbol 13-acetate, a PKC activator, disassembled the spheroids through epithelial-mesenchymal transition and increased their proliferation rate, while inducing cell death under monolayer culture conditions. These unusual behaviors of endometrial epithelial cells with collagen fibers could be a target for the treatment of some endometrial diseases.

Sorafenib targets and inhibits the oncogenic properties of endometrial cancer stem cells via the RAF/ERK pathway

BackgroundDistinct subsets of cancer stem cells (CSCs) drive the initiation and progression of malignant tumors via enhanced self-renewal and development of treatment/apoptosis resistance. Endometrial CSC-selective drugs have not been successfully developed because most endometrial cell lines do not contain a sufficient proportion of stable CSCs. Here, we aimed to identify endometrial CSC-containing cell lines and to search for endometrial CSC-selective drugs.MethodsWe first assessed the presence of CSCs by identifying side populations (SPs) in several endometrial cancer cell lines. We then characterized cell viability, colony-formation, transwell invasion and xenotransplantion capability using the isolated SP cells. We also conducted real-time RT-PCR, immunoblot and immunofluorescence analyses of the cells’ expression of CSC-associated markers. Focusing on 14 putative CSC-selective drugs, we characterized their effects on the proliferation and apoptosis of endometrial cancer cell lines, examining cell viability and annexin V staining. We further examined the inhibitory effects of the selected drugs, focusing on proliferation, invasion, expression of CSC-associated markers and tumor formation.ResultsWe focused on HHUA cells, an endometrial cancer cell line derived from a well-differentiated endometrial adenocarcinoma. HHUA cells contained a sufficient proportion of stable CSCs with an SP phenotype (HHUA-SP). HHUA-SP showed greater proliferation, colony-formation, and invasive capabilities compared with the main population of HHUA cells (HHUA-MP). HHUA-SP generated larger tumors with higher expression of proliferation-related markers, Ki67, c-MYC and phosphorylated ERK compared with HHUA-MP when transplanted into immunodeficient mice. Among the 14 candidate drugs, sorafenib, an inhibitor of RAF pathways and multiple kinase receptors, inhibited cell proliferation and invasion in both HHUA-SP and -MP, but more profoundly in HHUA-SP. In vivo treatment with sorafenib for 4 weeks reduced the weights of HHUA-SP-derived tumors and decreased the expression of Ki67, ZEB1, and RAF1.ConclusionsOur results suggest that HHUA is a useful cell line for discovery and identification of endometrial CSC-selective drugs, and that sorafenib may be an effective anti-endometrial cancer drug targeting endometrial CSCs.

High androgen level causes recurrent miscarriage and impairs endometrial receptivity

Purpose: To investigate the effect of androgen (A2) at different concentrations on human endometrial epithelial cancer cell line (HHUA) using iTRAQ and liquid chromatography–mass spectrometry (LCMS/MS) analysis.
 Methods: Human endometrial cells were cultured in Dulbecco Modified Eagle Medium. Total protein was isolated from human endometrial cells and HHUA cells were treated with A2 in three different concentrations (10-9 , 10-8 and 10-7 M). The proteins were digested using filter-aided sample preparation (FASP) method, and labeled using iTRAQ. The proteins were then subjected to LC-MS/MS. The resultant peptides were identified using Expasy tool, and the up-stream and down-stream proteins were confirmed. Moreover, the up-regulated proteins: isoform 2 of nuclear ubiquitous casein and cyclindependent kinase substrate 1 (NUCKS1), isoform 2 of endothelial differentiation-related factor 1 (EDF1), and acid phosphatase 1 (ACP1) were independently confirmed using western blotting and immunohistochemistry techniques.
 Results: Transforming growth factor (TGF), p38 mitogen-activated protein kinase (p38-MAPK), cyclindependent kinase (CDK), and tumor protein p53 (TP53) were the major regulators of the upstream process, and were upregulated RM-linked endometrial epithelial cells. Androgen-associated proteins NUCKS1, EDF1, and ACP1 were significantly responsible for miscarriage-related changes in endometrial tissues (p < 0.05). The results showed that a high level of androgen significantly (p < 0.05) distorts the expression levels of proteins associated with endometrium development, resulting in impaired endometrial accessibility which led to miscarriage.
 Conclusion: Androgen-associated proteins are responsible for changes in endometrial tissues which, in turn, lead to recurrent miscarriage.

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.

MiR‑494‑3p promotes the progression of endometrial cancer by regulating the PTEN/PI3K/AKT pathway.

MicroRNAs (miRs) are essential regulators in the development and progression of cancer. The role of miR-494-3p in endometrial cancer (EC) has not yet been investigated. In the present study, the expression levels of miR‑494‑3p were significantly upregulated in EC tissues compared with adjacent normal tissues. Furthermore, upregulation of miR‑494‑3p in patients with EC indicated poorer prognosis; miR‑494‑3p overexpression significantly promoted the proliferation, migration and invasion of HHUA and JEC cells invitro. Consistently, inhibition of miR‑494‑3p in HHUA cells significantly suppressed tumor growth invivo in a xenograft model. Additionally, phosphatase and tensin homolog(PTEN) was revealed to be a direct target of miR‑494‑3p in EC cells. Furthermore, overexpression of miR‑494‑3p inhibited PTEN expression and consequently activated the downstream phosphoinositide3‑kinase/protein kinaseB (PI3K/AKT) signialing pathway. Restoration of PTEN or inhibition of PI3K/AKT pathway also abolished miR‑494‑3p‑mediated proliferation, migration and invasion of HHUA and JEC cells. In summary, the results of the present study revealed the importance of the miR‑494‑3p/PTEN/PI3K/AKT axis in the progression of EC, which may provide novel insight into potential therapeutic targets for the treatment of EC.

Mifepristone inhibits proliferation, migration and invasion of HUUA cells and promotes its apoptosis by regulation of FAK and PI3K/AKT signaling pathway.

PurposeThe aim was to investigate mifepristone effects on endometrial carcinoma and the related mechanism.MethodsHHUA cells were treated with DMEM containing different concentrations of mifepristone. HHUA cells treated with 100 μmol/L mifepristone were named the Mifepristone group. HHUA cells co-transfected with pcDNA3.1-PI3K and pcDNA3.1-AKT overexpression vectors were treated with 100 μmol/L mifepristone and named the Mifepristone + PI3K/AKT group. mRNA expression was detected by quantitative reverse transcription PCR. Protein expression was performed by Western blot. Cell proliferation was conducted by MTT assay. Wound-healing assay was conducted. Transwell was used to detect cells migration and invasion. Apoptosis detection was performed by flow cytometry.ResultsMifepristone inhibited HHUA cells proliferation in a dose-dependent manner. Compared with HHUA cells treated with 0 μmol/L mifepristone, HHUA cells treated by 50–100 μmol/L mifepristone had a lower wound-healing rate, a greater number of migrating and invasive cells (P<0.01), as well as a higher percentage of apoptotic cells and Caspase-3 expression (P<0.01). When HHUA cells were treated with 50–100 μmol/L of mifepristone, FAK, p-FAK, p-PI3K and p-AKT relative expression was all significantly lower than HHUA cells treated with 0 μmol/L of mifepristone (P<0.01). Compared with the Mifepristone group, HHUA cells of the Mifepristone + PI3K/AKT group had a lower cell growth inhibition rate and percentage of apoptotic cells (P<0.01).ConclusionMifepristone inhibited HUUA cells proliferation, migration and invasion and promoted its apoptosis by regulation of FAK and PI3K/AKT signaling pathway.

Ramelteon, a selective MT1/MT2 receptor agonist, suppresses the proliferation and invasiveness of endometrial cancer cells

The incidence of endometrial cancer is increasing, making it the fifth most common cancer worldwide. To date, however, there is no standard therapy for patients with recurrent endometrial cancer. Melatonin, a hormone secreted by the pineal gland, has been shown to have anti-tumor effects in various tumor types. Although melatonin is available as a supplement, it has not been approved for cancer treatment. Ramelteon, a selective melatonin receptor type 1 and 2 (MT1/MT2) receptor agonist, has been approved to treat sleep disorders, suggesting that ramelteon may be effective in the treatment of endometrial cancer. To determine whether this agent may be effective in the treatment of endometrial cancer, this study investigated the ability of ramelteon to suppress the proliferation and invasiveness of HHUA cells, an estrogen receptor-positive endometrial cancer cell line. Ramelteon at 10-8 M maximally suppressed the proliferation of HHUA cells, reducing the percentage of Ki-67 positive proliferating cells. This effect was completely blocked by luzindole, a MT1/MT2 receptor antagonist. Furthermore, ramelteon inhibited HHUA cell invasion and reduced the expression of the MMP-2 and MMP-9 genes. These results suggested that ramelteon may be a candidate for the treatment of recurrent endometrial cancer, with activity similar to that of melatonin.

Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells.

The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor β, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower β-tubulin levels and comparatively higher p53/β-tubulin or CAR /β-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

Sirtuin 1 promotes the growth and cisplatin resistance of endometrial carcinoma cells: a novel therapeutic target

Sirtuin 1 (SIRT1), originally identified as a longevity gene, is induced by caloric restriction, and regulates various cellular functions including DNA repair, cell survival and metabolism via the deacetylation of target proteins such as histone and p53. These functions are considered to act dualistically as preventing or facilitating cancer. This study aimed to clarify the expression and role of SIRT1 in endometrial carcinoma. Because a high-calorie diet was a well-known risk factor for endometrial carcinoma, we first hypothesized that SIRT1 might be downregulated in normal endometrial glandular cells of obese women. However, no correlation was observed between the expression of SIRT1 and body mass index (BMI). In contrast, regardless of BMI, the immunohistochemical expression of SIRT1 was significantly higher in endometrial carcinoma (108 cases) than in normal endometria (60 cases) (P<0.05), and its overexpression was associated with a shorter survival (P<0.05). Our experiments in vivo revealed that SIRT1 accelerated the proliferation of endometrial carcinoma cell lines (HHUA, HEC151, and HEC1B). SIRT1 overexpression significantly enhanced the resistance for cisplatin and paclitaxel in HHUA cells. Although p53 is an important target protein for SIRT1, the selective SIRT1 inhibitor (EX527) significantly suppressed the proliferation and cisplatin resistance of three endometrial carcinoma cell lines regardless of the p53 mutation status. In addition, SIRT1 overexpression in HHUA cells accelerated tumor growth and cisplatin resistance in nude mice, and EX527 significantly suppressed the growth of tumors of HHUA and HEC1B cells. No adverse effect of EX527 was observed in these mice. In conclusion, SIRT1 is involved in the acquisition of the aggressive behavior associated with endometrial carcinoma, and the SIRT1 inhibitor, EX527, may be a useful agent for the treatment of this malignancy.

Telmisartan Induces Growth Inhibition, DNA Double-Strand Breaks and Apoptosis in Human Endometrial Cancer Cells

Telmisartan, an angiotensin II receptor type 1 blocker, is often used as an antihypertension drug, and it has also been characterized as a peroxisome proliferator-activated receptor-gamma (PPARγ) ligand. The purpose of this study was to elucidate the antitumor effects of telmisartan on endometrial cancer cells. We treated three endometrial cancer cell lines with various concentrations of telmisartan, and we investigated the effects of the telmisartan on the cell proliferation, apoptosis, and their related measurements in vitro. We also administered telmisartan to nude mice with experimental tumors to determine its in vivo effects and toxicity. All three endometrial cancer cell lines were sensitive to the growth-inhibitory effect of telmisartan. The induction of apoptosis was confirmed in concert with the altered expression of genes and proteins related to the apoptosis. We also observed that DNA double-strand breaks (DSBs) were induced in HHUA (human endometrial cancer) cells by telmisartan treatment. In addition, experiments in nude mice showed that telmisartan significantly inhibited human endometrial tumor growth, without toxic side effects. Our results suggest that telmisartan might be a new therapeutic option for the treatment of endometrial cancers.

Abstract 3046: Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell.

Abstract Background: The mechanism of carcinogenesis and progression of endometrial carcinoma has not been fully understood. To elucidate the molecular machinery, we performed microarray analysis using mRNA extracted from normal endometria, endometrial hyperplasia and carcinoma and found lipocalin2 (LCN2), an iron transporter, to be markedly up-regulated along with disease progression. Furthermore, the over-expression of LCN2 and its receptor, SLC22A17, was associated with poor outcome of the patients with endometrial carcinoma. In the present study, we further analyzed the molecular functions of LCN2 using endometrial carcinoma cells. Methods: An endometrial carcinoma cell line HHUA over-expressing both LCN2 and SLC22A17 was used. We established LCN2-silenced HHUA (HHUAsh) and control-HHUA (HHUAc) by transfection of LCN2-shRNA and scrambled shRNA producing vector, respectively. The growth and migration were examined by WST-1 assay and wound healing assay. Cell viability under stress conditions such as ultraviolet (UV) irradiation or cisplatin (CDDP) treatment, with or without iron-chelation, was measured by WST-1. The intensity of apoptosis was analyzed by TUNEL staining and Apostrand assay. The expression of phosphorylated Akt was examined by Western blotting. In vivo tumor formation was observed by the subcutaneous injection of those HHUA cells to nude mice. The colony formation of these HHUA cells in soft agar was evaluated. In addition, the LCN2 cDNA containing retrovirus vector (LCN2-RET) was transfected to NIH3T3 to observe the transformation ability. Results: The cell migration of HHUAsh was decreased compared with HHUAc. After UV irradiation and CDDP treatment, the residual cell viability of HHUAsh (25.4% and 37.1%, respectively) was significantly lower than that of HHUAc (51.2% and 77.6%, p&amp;lt;0.05). Under these stresses, the enhanced apoptosis and down-regulation of phosphorylated Akt was observed in HHUAsh. Interestingly, these differences were abolished by iron-chelation. The growth of HHUAsh showed no difference with that of HHUAc in vitro. However, LCN2-silencing significantly reduced the tumor growth in nude mice (84% reduction). Moreover, the down-regulation of phospho-Akt expression was observed in tumors of LCN2-silenced HHUA. The colony formation in soft agar of HHUAsh was significantly reduced (66.3% reduction) from that of HHUAc, and this difference was rescued to 12% by addition of recombinant LCN2. In addition, NIH3T3 transfected by LCN2-RET formed focuses. Conclusions: The present study revealed that LCN2 is involved in the genesis and survival of endometrial carcinoma cells under stress conditions and in the tumor growth in vivo, via p-Akt signaling in an iron-dependent manner, suggesting LCN2 to be a novel oncogene. Citation Format: Tsutomu Miyamoto, Ryoichi Asaka, Yasushi Yamada, Kaori Ishikawa, Hisanori Kobara, Akihisa Suzuki, Tanri Shiozawa. Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2013-3046

GRP78 induced by estrogen plays a role in the chemosensitivity of endometrial cancer

ObjectiveMolecular chaperone 78kDa glucose-regulated protein (GRP78) is a residential protein in the endoplasmic reticulum (ER) that is induced by an unfolded-protein response triggered under many kinds of stress against a cell. GRP78 is also known to act as an anti-apoptotic factor by protecting ER-stress-induced cell death. In this study, we examined the significance of GRP78 expression in endometrial cancer. MethodsTissue samples obtained from patients with a diagnosis of enodometrial cancer were subjected to immunohistochemistry and RT-PCR to determine protein and mRNA expression levels of GRP78 and estrogen receptor α. We used Western blot and RT-PCR to examine whether estrogen induced GRP78 expression in cancer cell lines. Western blots and MTT assays of GRP78 siRNA transfected Ishikawa and HHUA cells were used to demonstrate whether GRP78 is involved in chemoresistence. ResultsGRP78 was highly expressed in well and moderately differentiated endometrial carcinoma. Estrogen induced GRP78 expression, which was correlated with cell viability and resistance to paclitaxel and cisplatin. Western blot analysis indicated that active caspase-3 and the 85-kDa protein poly (ADP-ribose) polymerase (PARP) were increased by incubation with either paclitaxel or cisplatin, suggesting that the apoptotic pathway was involved in cancer-drug-induced cell death. ConclusionsThese results may open up a novel therapeutic strategy for endometrial cancer: namely, the targeting of GRP78 to sensitize the tumor cell to chemotherapy.

MSH3 Mediates Sensitization of Colorectal Cancer Cells to Cisplatin, Oxaliplatin, and a Poly(ADP-ribose) Polymerase Inhibitor

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.

STAT3 enhances intracellular Fas-mediated apoptotic signals in HHUA human endometrial epithelial cells

Several endometrial signal transducer and activator of transcription 3 (STAT3)-activating cytokines are reported to be essential for blastocyst implantation, with inhibition of STAT3 activation in the endometrium also reported to prevent implantation. To investigate STAT3 signals in endometrial epithelial cells, the activation and inactivation effects of STAT3 signals were examined in the human endometrial epithelial cell line HHUA, which is thought to retain many of the intracellular signaling pathways found in normal human endometrial epithelial cells. Five STAT3-activating cytokines, IL-11, IL-10, LIF, oncostatin M and leptin, enhanced the Fas-mediated apoptosis of the HHUA cells without any increase in cell surface Fas antigen expression. STAT3 siRNA transfection suppressed STAT3 expression in HHUA cells and significantly inhibited Fas-mediated cell death. These results indicate that intracellular apoptotic signals in HHUA cells are constitutively activated and regulated by STAT3-mediated signals. This apoptosis-promoting effect of STAT3 in HHUA cells is completely different from many previous reports demonstrating anti-apoptotic effects by STAT3 activation. The STAT3 signals in HHUA cells may be specific to the human endometrial epithelial cell lineage in the regulation of blastocyst implantation.

Suppressed protein expression of the death-associated protein kinase enhances 5-fluorouracil-sensitivity but not etoposide-sensitivity in human endometrial adenocarcinoma cells

Targeted knockdown of the death-associated protein kinase (DAPK) expression in the endometrial adenocarcinoma HHUA cells reportedly induces cell death by enhancing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in an autocrine/paracrine manner. This suggests that endogenous DAPK is a potential candidate for a molecularly targeted anticancer therapy for patients with endometrial adenocarcinoma. To investigate the role of endogenous DAPK in anticancer drug sensitivity, we examined effects on cellular anticancer drug sensitivities of transfections with 5 different specific DAPK small-interfering RNAs (siRNAs) into HHUA cells. DAPK siRNA transfections strongly enhanced 5-fluorouracil (5FU)-sensitivity, but not etoposide-sensitivity, of HHUA cells compared with control siRNA-transfected cells. These results indicate that etoposide-stimulated cell death signals may share or include TRAIL-mediated apoptotic signals, and that 5FU-stimulated cell death signals may be independent from TRAIL-mediated apoptotic signals induced by DAPK siRNA transfections. Moreover, 5FU-combined chemotherapy with DAPK siRNA transfection may show stronger anticancer effects on patients with endometrial adenocarcinoma than does chemotherapy alone.

Death-associated protein kinase is essential for the survival of various types of uterine cancer cells

We recently showed that targeted knockdown of death-associated protein kinase (DAPK) expression induces apoptosis in the human endometrial adenocarcinoma cell line HHUA. To investigate the possibility that DAPK may represent a molecular target for anticancer therapies for advanced uterine cancers, we examined the effects of DAPK siRNA transfections on the viability of five different human uterine cancer cell lines. The five uterine cell lines comprised three differentiated endometrial adenocarcinomas, one leiomyosarcoma and one carcinosarcoma. Cell death assays showed that the DAPK siRNA transfection significantly increased the cell death in all five uterine cancer cells examined. Ribonuclease protection assays did not show any remarkable changes in the bcl-2 family gene expressions after the DAPK siRNA transfection in HHUA cells. Since DAPK-mutant mice were reported to be fertile and do not show lethality, DAPK may play a central role in the immortalization and carcinogenesis of uterine cancer cells, possibly without bcl-2 family-related apoptotic regulation. These results indicate that DAPK can be a convincing candidate for molecularly targeted anticancer therapies for patients with various types of advanced uterine cancers, including carcinosarcoma and leiomyosarcoma.

Targeted knockdown of death-associated protein kinase expression induces TRAIL-mediated apoptosis in human endometrial adenocarcinoma cells

Death-associated protein kinase (DAPK) is a serine/threonine kinase that participates in the modulation of apoptosis and tumor suppression. Our previous study revealed high levels of DAPK protein expression in differentiated endometrial adenocarcinoma cells. To clarify the role of DAPK in human endometrial adenocarcinomas, we down-regulated endogenous DAPK expression in HHUA cells, a well-differentiated endometrial adenocarcinoma cell line, using specific small-interfering RNAs (siRNAs). The suppression of endogenous DAPK expression triggered apoptosis in HHUA cells, as evidenced by an increase in the sub-G1 DNA content in flow cytometric analyses. The apoptosis induced by the DAPK siRNA transfections was caspase-dependent, as characterized by the activations of caspase-3, -8 and -9. RNase protection assays detected higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 transcripts in the DAPK siRNA-transfected HHUA cells than in the control siRNA-transfected cells. Consistent with these findings, enzyme-linked immunosorbent assays revealed that the DAPK siRNA transfections significantly increased the secretion of TRAIL protein from the cells. Treatment with recombinant human TRAIL protein dose-dependently suppressed the cell viability of HHUA cells. The present findings reveal that down-regulation of endogenous DAPK expression in HHUA cells induces caspase-dependent apoptosis, possibly through increased TRAIL, DR4 and DR5 signaling, thereby suggesting that DAPK expression is essential for HHUA cell survival. Consequently, endogenous DAPK mRNA may represent a potential candidate for molecularly targeted anticancer therapies.

The effect of cinobufagin on the expression of survivin of HHUA cells in endometrial carcinoma

Objective To investigate the effect and significance of cinobufagin on the expression of survivin of HHUA cells in the endometrial carcinoma cultured in vitro. Methods HHUA cells of endometrial carcinoma were cultured in vitro. After being intervened by cinobufagin with different concentration, the expression of survivin was measured by RT-PCR and the cellular apoptosis was tested by flow cytometry. Results In the control group: mRNA expression of survivin of HHUA cells in endometrial carcinoma was (1.22±0.23), cellular apoptutic rate was (2.31± 0.98)%; cinobufagin of 0.25 mg/ml didn't affect the survivin and cellular apoptotic rate (q=2.89, P>0.05: q=3.12, P>0.05) ; after being exposed to einobufagin of 2.5 mg/ml, the expression of survivin was (0.73± 0.26), and cellular apoptotic rate was (26.50± 6.36) %, with all of these data being different from the control group significantly (q=5.68, P 0.05; q=4.35, P>0.05) . Conclusion Cinobufagin of 2.5 mg/ml inhibit the mRNA expression of survivin and proliferation of HHUA cells in endometrial carcinoma. Key words: Cinobufagin; Endometrialcarcinoma; Survivin; Apoptosis