Apoptosis In Endometrial Cancer Cells Research Articles

Cisplatin increases B‐cell‐lymphoma‐2 expression via activation of protein kinase C and Akt2 in endometrial cancer cells

Human carcinomas often show resistance to cisplatin and Bcl-2 is associated with resistance to cisplatin. However, Bcl-2 regulation on cisplatin treatment in human cancers is unknown. Here, we show a novel mechanism by which cisplatin treatment promotes resistance by increasing the expression of Bcl-2 mRNA. Bcl-2 mRNA and protein expression was increased in cisplatin-resistant endometrial cancer cell lines (KLE and HEC-1-A), but not in cisplatin-sensitive cell line (Ishikawa). Cisplatin-mediated increase in Bcl-2 expression was blocked by combination with either actinomycin D or cycloheximide. In addition, Bcl-2 inhibition by HA14-1 led to increased cisplatin-induced apoptosis in KLE and HEC-1-A, whereas Bcl-2 overexpression in Ishikawa led to decreased cisplatin-induced apoptosis. Inhibition of protein kinase C (PKC) activity prevented cisplatin-dependant increase in Bcl-2 mRNA, and induced apoptosis in KLE cells. Furthermore, PKC inhibition was associated with decreased Akt and NF-κB activity. Cells stably expressing shRNA for Akt isoforms revealed that Akt2 was involved in cisplatin-dependant increase in Bcl-2 and apoptosis. Overexpression of Akt2 in Akt2-deficient cells led to increased Bcl-2 expression on cisplatin treatment. Our data suggest a novel regulation pathway of Bcl-2 by cisplatin, via the activation of PKC and Akt2, which has a profound impact on resistance to cisplatin-induced apoptosis in endometrial cancer cells.

Abstract 1107: Transforming growth factor-beta (TGF-β) increases Par-4 expression via activation of Smad and NF-κB in endometrial cancer cells

Abstract Prostate apoptosis response-4 (Par-4) gene was originally identified in prostate cancer cells undergoing apoptosis. Further studies have shown that Par-4 selectively induces apoptosis in cancer cells without effecting normal cells. Recently it has been evidenced that Par-4 has a tumor suppressor role in endometrial cancer. We have previously demonstrated that TGF-β isoforms are expressed in endometrial tumors and determined cellular fate by regulating apoptosis through the activation of NF-κB. In the present study, we have investigated the mechanisms through which TGF-β isoforms regulate Par-4 expression in endometrial cancer cells. KLE and Hec-1-A endometrial cancer cell lines were used as model in this study. Transcript and protein levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting, respectively. The results revealed that both cell lines express TGF-β receptor I and receptor II. Blocking TGF-β signalling by neutralizing TGF-β antibody reduced endogenous Par-4 mRNA and protein levels. Exogenous treatment with all the TGF-β isoforms (10ng/ml) upregulated Par-4 mRNA and protein levels. Furthermore, there was increase in the levels of phosphorylated Smad2 and Smad3 in response to TGF-β treatment, thus indicating that Smad pathway was activated by TGF-β isoforms in these cells. However, there was no change in the protein levels of total Smad2/3. TGF-β treatment resulted in rapid phosphorylation of IκB-α with no effect on total IκB-α levels thus suggesting an activation, nuclear translocation and increase in transcriptional activity of NF-κB. Collectively, these results suggest that each TGF-β isoform induces Par-4 levels via the activation of Smad and NF-κB pathway in endometrial cancer cells. Since downregulation of Par-4 is a common event in different types of cancers, we suggest in these cases that TGF-β could be an interesting therapy and strategy to induce Par-4 and apoptosis in endometrial cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1107. doi:10.1158/1538-7445.AM2011-1107

Function of UDP-glucuronosyltransferase 2B17 (UGT2B17) is involved in endometrial cancer

Endometrial cancer (EC) is a steroid hormone-dependent cancer. Uridine 5'-diphospho-glucuronosyltransferase enzymes conjugate and detoxify endogenous and exogenous steroid hormones and environmental carcinogens. Among these enzymes, the function of UGT2B17 is unknown except for glucuronidation. The messenger RNA expression of UGT2B17 and myeloid cell leukemia-1 (Mcl-1) was significantly increased in EC tissues compared with matched normal endometrial tissues. Therefore, we focused on the function of UGT2B17 in EC. A total of nine patients with confirmed EC were enrolled in this study to investigate the expression of UGT2B17 and target genes. EC cell lines were used for functional tests including cell growth, invasion, apoptosis and cell cycle analyses. To find the target genes of UGT2B17, we performed microarray analysis to see which genes were upregulated or downregulated by UGT2B17-transfected cells. Functional analysis showed decreased numbers of viable cells and increased numbers of apoptotic cells in si-UGT2B17-transfected Ishikawa cells. Among microarray target genes, Mcl-1 was significantly downregulated in si-UGT2B17-transfected cells. We also found upregulation of Puma protein, a target of Mcl-1, in si-UGT2B17-transfected cells. This is the first report to show that UGT2B17 and Mcl-1 expression are upregulated in EC tissues and that UGT2B17 depletion induces inhibition of cell growth and apoptosis in EC cells through Mcl-1 downregulation.

Expression of glyoxalase I and its effect on cell proliferation and apoptosis in endometrial carcinoma

To examine the expressions of glyoxalase I (GLO-I) in endometrial cancer tissues and cell lines and to investigate the roles of GLO-I on proliferation and apoptosis in endometrial cancer cells. Immunohistochemistry, western blot and RT-PCR were used to investigate the expressions of GLO-I protein and mRNA in endometrial cancer tissues and Ishikawa cell lines;enzyme activity of GLO-I in normal endometrium, endometrial cancer and paraneoplastic tissue samples was detected with spectrophotometer;proliferation and apoptosis of Ishikawa cell before and after RNA interference (RNAi) procedure were detected by the methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. (1) There were significant differences of GLO-I expression between normal endometrium (0/19) and endometrial cancer tissues (76%, 22/29); these were also significant differences of enzyme activity of GLO-I among normal endometrium, paraneoplastic and endometrial cancer tissues (1.1, 0.8 vs 92.3 IU/mg; P < 0.01). Enzyme activity of GLO-I in fresh normal endometrium and paraneoplastic tissues was weak, while that of fresh endometrial cancer tissues was as high as 92.3 IU/mg in average. (2) The expression of GLO-I mRNA in Ishikawa cell transfected with GLO-I siRNA was significantly lower than that in negative group (0.25 ± 0.06 vs 0.93 ± 0.10, P < 0.01), and the similar results that in the expression of GLO-I protein (0.38 ± 0.06 vs 0.94 ± 0.13, P < 0.01). (3) Proliferation in Ishikawa cell was significantly inhibited after silencing RNA expression of GLO-I (P = 0.028). The apoptosis rate of cells transfected with GLO-I siRNA was significantly higher than that of negative control group and blank control group [(6.7 ± 0.8) % vs (1.2 ± 0.4)%, (1.4 ± 0.4)%; P < 0.01]. The expression and enzyme activity of GLO-I is significantly increased in endometrial cancer, which could promote abnormal proliferation and inhibit apoptosis in endometrial cancer cells.

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

Background Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established. Scope of Review EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells. Major Conclusions Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways. General significance These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.

Abstract 1196: Progesterone via the PRB receptor attenuates AKT inhibitor mediated apoptosis in endometrioid endometrial cancer

Abstract Mutations in the PTEN gene are the most common genetic defects in endometrioid endometrial carcinoma and seen in more than 50% of tumors. PTEN, a tumor suppressor gene, negatively regulates PI3K/Akt-driven cell growth and survival. Previously, we have shown that levels of the forkhead protein, FOX01, a direct target of AKT, were significantly lower in endometrial carcinoma and overexpression of this gene in endometrial cancer cell lines resulted in decreased cell proliferation and increased apoptosis. Interestingly, the function of FOX01 in this context was determined by the progesterone receptor isoform that was expressed, strongly implicating the regulatory role of the progesterone receptor predictably at the transcription level. In this study, we aimed to look at the effects of an AKT inhibitor (APi59) on endometrial cancer cells and the influence of PRA or PRB on its efficacy. The cell viability assay revealed a decrease in the number of viable PRA and PRB cells treated with APi59 for 48 hours, while the progestin, R5020, alone did not affect cell viability. In addition, more cell death was observed in the PRA cells compared to the PRB cells. APi59 treatment promoted apoptosis, as measured by cleaved PARP levels, in both PRA and PRB cells, with increased apoptosis in PRA cells compared to the PRB cells. R5020 treatment alone did not cause apoptosis as compared to control. In order to further investigate the increased levels of apoptosis in the PRA cells, a real time PCR array focused on genes associated with apoptosis was used to identify potential genes involved in this differential response. The most highly upregulated gene specifically expressed in PRB cells in response to R5020 was BIRC3 (ciap2). BIRC3 was upregulated 13-fold with R5020 treatment in PRB cells where its expression in PRA cells was unaffected. Given that BIRC3 is a member of the inhibitor of apoptosis genes (IAP), we hypothesized that the increased expression of BIRC3 in PRB cells attenuated APi59-mediated apoptosis. Thus, PR was silenced using transient transfection of siRNA specific to PR and cells were treated with APi59 and R5020. Upon PR silencing, there was an increase in apoptosis as measured by cleaved PARP in APi59 treated cells along with a concomitant decrease in BIRC3 protein. To determine if the attenuation in apoptosis through BIRC3 occurred with other cytotoxic agents, PRB cells were treated with 10nm paclitaxel and R5020. Again, apoptosis in response to paclitaxel was attenuated, as shown by lower cleaved PARP levels when R5020 was added. In conclusion, not only can the AKT inhibitor, APi59, promote apoptosis in endometrial cancer cells, but liganded PRB can blunt the apoptotic response through increased expression of BIRC3, an inhibitor of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1196.

Sojucktang induces apoptosis via loss of mitochondrial membrane potential and caspase-3 activation in KLE human endometrial cancer cells

Sojucktang (SJT) has long been used for the treatment of endometrial diseases in Korea. However, the mechanisms responsible for the SJT-induced apoptosis in endometrial cancer cells remain unclear. In the present study, SJT was demonstrated to show cytotoxic effect and induce apoptotic cell death via mitochondrial regulation in KLE endometrial cancer cells. Linderae Radix, Glycyrrhizae Radix, Zedoariae Rhizoma, Trogopterorum Faeces and Agelicae Gigantis Radix were found to be the potent constituent herbs of SJT to significantly decrease the viability of KLE cells by a tetra zolium salt (XTT) assay. Apoptotic bodies were observed in SJT-treated KLE cells by 4′-6-diamidino-2-phenylindole (DAPI) and TdT-mediated-dUTP nick-end labeling (TUNEL) assay. SJT also increased sub-G1 DNA contents of the cell cycle undergoing apoptosis in a dose-dependent manner. Furthermore, it was observed that SJT activated caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP), and decreased mitochondrial membrane potential in a dose-dependent manner. Taken together, this study shows that SJT exerts anti-tumor activity against KLE endometrial cancer cells via mitochondrial dependent apoptosis induction.

Gonadotropin-Releasing Hormone Type II Induces Apoptosis of Human Endometrial Cancer Cells by Activating GADD45α

Gonadotropin-releasing hormone type II (GnRH-II) has an antiproliferative effect on human endometrial cancer cells. Apoptosis in cancer cells may play a critical role in regulating cell proliferation. However, more studies are necessary to elucidate the underlying molecular mechanisms and develop potential applications of GnRH-II. Therefore, we explored the mechanisms of GnRH-II-induced apoptosis and the effects of GnRH-II on GADD45alpha activation in human endometrial cancer cell lines. GnRH-II decreased cell viability in a dose- and time-dependent manner. Apoptosis was induced with increased terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling apoptotic cells after GnRH-II treatment. Knockdown of the endogenous GnRH-I receptor with small interfering RNA (siRNA) rescued the cells from GnRH-II-mediated cell growth inhibition and abolished the induction of apoptosis. GnRH-II activated extracellular signal-regulated kinase (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) in a time-dependent manner, and the activation was abolished by GnRH-I receptor siRNA and MAPK inhibitors. Cells pretreated with MAPK inhibitors were rescued from GnRH-II-mediated cell growth inhibition. Moreover, both inhibitors abolished GnRH-II-induced apoptosis. GnRH-II induced GADD45alpha expression, which was abolished by knockdown of endogenous GnRH-I receptors and MAPK inhibitors. GnRH-II-stimulated cell growth inhibition was rescued by knockdown of endogenous GADD45alpha with siRNA. Cells treated with GADD45alpha siRNA were refractory to GnRH-II-induced apoptosis. Thus, GnRH-II inhibits cell growth by inducing apoptosis through binding of the GnRH-I receptor, activation of the ERK1/2 and p38 MAPK pathways, and induction of GADD45alpha signaling. This finding may provide a new concept relating to the mechanism of GnRH-II-induced antiproliferation and apoptosis in endometrial cancer cells, indicating the possibility of GnRH-II as a promising therapeutic intervention for human endometrial cancer.

C-Jun N-terminal kinase regulates apoptosis in endometrial cancer cells

c-Jun N-terminal kinases (JNKs) are important regulators of cell proliferation and apoptosis that have been implicated in tumorigenesis. We investigated the role of JNKs in apoptotic responses in Ishikawa and HEC-50 cells, models of type I and type II endometrial cancer, respectively. Etoposide treatment or UV irradiation resulted in sustained activation of JNK, correlating with the induction of apoptosis. Inhibition of JNK, or MAP kinase kinase 4 (MKK4), selectively suppressed apoptotic responses in both Ishikawa and HEC-50 cells. Knockdown of protein kinase C delta (PKCdelta) also attenuated apoptosis in endometrial cancer cells and inhibited the sustained, UV-mediated JNK activation in HEC-50, but not Ishikawa cells. Etoposide-induced JNK phosphorylation was unaffected by PKCdelta knockdown, implying that JNK can regulate apoptosis by PKCdelta-dependent and independent pathways, according to stimulus and cell type. Thus, expression and activity of JNK and PKCdelta in endometrial cancer cells modulate apoptosis and sensitivity to chemotherapeutic agents and may function as tumor suppressors in the endometrium.

Induction of apoptosis in endometrial cancer cells by psammaplysene A involves FOXO1

Objective Endometrial cancer is the most common type of gynecologic cancer in the United States. In this study, we propose that a marine sponge compound, psammaplysene A (PsA) induces apoptosis in endometrial cancer cells through forced nuclear expression of FOXO1. Methods Ishikawa and ECC1 cells were treated with varying doses of PsA. FOXO1 protein localization was observed using immunofluorescent staining of cells. The effects of PsA on cell viability and proliferation were assessed using a cell viability assay and a BrdU incorporation assay respectively. Cell cycle analysis was performed using flow cytometry. To assess the role of FOXO1 in PsA-induced apoptosis, FOXO1 was silenced in ECC1 cells using siRNA technique, and overexpressed in Ishikawa cells using an adenovirus containing FOXO1 cDNAs. Western blots were used to measure levels of FOXO1 and cleaved PARP proteins. Results Treatment of both ECC1 and Ishikawa cells with PsA caused an increase in nuclear FOXO1 protein, a dramatic decrease in cell viability of approximately 5-fold ( p < 0.05) and minimal effect on proliferation. Furthermore, treatment of cells with PsA doubled the percentage of cells in the G2/M phase ( p < 0.05). PsA induced apoptosis in endometrial cancer cells. When FOXO1 was silenced in ECC1 cells and treated with PsA, the incidence of apoptosis decreased. In addition, overexpression of FOXO1 with PsA treatment increased apoptosis. Conclusions Increasing nuclear FOXO1 function is important for the induction of apoptosis of endometrial cancer cells by PsA.

Raloxifene, a selective estrogen receptor modulator, induces mitochondria-mediated apoptosis in human endometrial carcinoma cells

Raloxifene is a nonsteroidal benzothiophene that has also been classified as a selective estrogen receptor modulator (SERM) on the basis of studies in which it produced both estrogen-agonistic effects on bone and lipid metabolism and estrogen-antagonistic effects on uterine endometrium and breast tissue. We investigated apoptotic cell death and the apoptotic pathway in human endometrial carcinoma cells (Ishikawa cells) expressing estrogen receptor treated with raloxifene. Cell viability was significantly decreased in Ishikawa cells treated with raloxifene at 20 microM and higher levels. Raloxifene at 20 microM induced 54% inhibition of cell viability after 48 h treatment. Apoptotic parameters were analyzed for determination of apoptotic pathway in Ishikawa cells treated with 20 microM or 40 microM raloxifene for 48 h. The numbers of apoptotic cells were significantly increased in cells treated with raloxifene as compared with control cells. Activities of caspase-3,-8, and-9 were significantly elevated in Ishikawa cells treated with raloxifene. A significant decrease in mitochondrial membrane potential was observed in this treatment. In addition, the levels of cytosolic cytochrome c were significantly elevated in raloxifene-treated cells. Expression of Bid was detected in both control and raloxifene-treated cells, but Bid cleavage was not observed. In caspase inhibitor experiments, cell viability was significantly increased by the caspase-9 inhibitor z-LEHD-fmk and by the caspase-3 inhibitor z-DEVD-fmk. However, cell viability was unaffected by addition of the caspase-8 inhibitor z-IETD-fmk. Thus, raloxifene induced mitochondria-mediated apoptosis in endometrial cancer cells but not via the Bid-mitochondria pathway. It is possibly that raloxifene may be useful as an adjuvant to current chemotherapies for endometrial cancer and possibly is useful as a chemopreventive agent.

Dichloroacetate induces apoptosis in endometrial cancer cells

Purpose A recent landmark study demonstrated that Dichloroacetate (DCA) treatment promoted apoptosis in lung, breast, and glioblastoma cancer cell lines by shifting metabolism from aerobic glycolysis to glucose oxidation coupled with NFAT-Kv1.5 axis remodeling. The objective of this study was to determine whether DCA induces apoptosis in endometrial cancer cells and to assess apoptotic mechanism. Methods A panel of endometrial cancer cell lines with varying degrees of differentiation was treated with DCA and analyzed for apoptosis via flow cytometry. Biological correlates such as gene expression, intracellular Ca 2+, and mitochondrial membrane potential were examined to assess apoptotic mechanism. Results Initiation of apoptosis was observed in five low to moderately invasive cancer cell lines including Ishikawa, RL95-2, KLE, AN3CA, and SKUT1B while treatment had no effect on non-cancerous 293T cells. Two highly invasive endometrial adenocarcinoma cell lines, HEC1A and HEC1B, were found to be resistant to DCA-induced apoptosis. Apoptotic responding cell lines had a significant increase in early and late apoptotis, a decrease in mitochondrial membrane potential, and decreased Survivin transcript abundance, which are consistent with a mitochondrial-regulated mechanism. DCA treatment decreased intracellular calcium levels in most apoptotic responding cell lines which suggests a contribution from the NFAT-Kv1.5-mediated pathway. DCA treatment increased p53 upregulated modulator of apoptosis (PUMA) transcripts in cell lines with an apoptotic response, suggesting involvement of a p53-PUMA-mediated mechanism. Conclusions Dichloroacetate effectively sensitizes most endometrial cancer cell lines to apoptosis via mitochondrial, NFAT-Kv1.5, and PUMA-mediated mechanisms. Further investigation of the cancer therapeutic potential of DCA is warranted.

Endometrial cancer cell survival and apoptosis is regulated by protein kinase C α and δ

Endometrial cancer is the most common invasive gynecologic malignancy but the molecular mechanisms underlying its onset and progression are poorly understood. Paradoxically, endometrial tumors exhibit increased apoptosis, correlating with disease progression and poor patient prognosis. Endometrial tumors also show altered activity and expression of protein kinase C (PKC) isoforms, implicated in the regulation of programmed cell death; however, PKC modulation of apoptosis in endometrial cancer cells has not been investigated. We detected nine out of ten PKC isoforms in Ishikawa endometrial cancer cell lines, and demonstrated expression of both PKCalpha and delta in human endometrial tumors. To determine the functional roles of PKCalpha and delta in apoptosis in endometrial cancer, Ishikawa cells were treated with selective PKC inhibitors or adenoviral constructs encoding wild-type or isoform-specific, dominant-negative mutants. Apoptosis was assessed by DNA fragmentation and caspase-mediated poly-(ADP-ribose)-polymerase cleavage. The inhibition of PKCdelta suppressed etoposide-induced apoptosis, while overexpression of PKCdelta enhanced it. In contrast, inhibition of PKCalpha elevated basal levels of apoptosis and potentiated etoposide-induced cell death. Etoposide treatment also selectively activated PKCdelta, but resulted in both cytosolic translocation and decreased activity of PKCalpha. A fraction of PKCdelta also underwent caspase-dependent cleavage, in response to etoposide. Our results suggest that changes in apoptosis and PKC expression in endometrial cancer are mechanistically linked, such that PKCdelta is required for DNA damage-induced apoptosis, while PKCalpha mediates a survival response. Thus, PKCalpha and delta expression and signaling may be important in endometrial tumorigenesis and could serve as potential prognostic indicators and/or novel targets for therapeutic intervention.