SUMO1 degrader induces ER stress and ROS accumulation through deSUMOylation of TCF4 and inhibition of its transcription of StarD7 in colon cancer.

Small molecule degraders of small ubiquitin-related modifier 1 (SUMO1) induce SUMO1 degradation in colon cancer cells and inhibits the cancer cell growth; however, it is unclear how SUMO1 degradation leads to the anticancer activity of the degraders. Genome-wide CRISPR-Cas9 knockout screen has identified StAR-related lipid transfer domain containing 7 (StarD7) as a critical gene for the degrader's anticancer activity. Here, we show that both StarD7 mRNA and protein are overexpressed in human colon cancer and its knockout significantly reduces colon cancer cell growth and xenograft progression. The treatment with the SUMO1 degrader lead compound HB007 reduces StarD7 mRNA and protein levels and increases endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in colon cancer cells and three-dimensional (3D) organoids. The study further provides a novel mechanism of the compound anticancer activity that SUMO1 degrader-induced decrease of StarD7 occur through degradation of SUMO1, deSUMOylation and degradation of T cell-specific transcription 4 (TCF4) and thereby inhibition of its transcription of StarD7 in colon cancer cells, 3D organoids and patient-derived xenografts (PDX).

Bioinformatics analyses of combined databases identify shared differentially expressed genes in cancer and autoimmune disease.

BACKGROUND: Inadequate immunity caused by poor immune surveillance leads to tumorigenesis, while excessive immunity due to breakdown of immune tolerance causes autoimmune genesis. Although the function of immunity during the onset of these two processes appears to be distinct, the underlying mechanism is shared. To date, gene expression data for large bodies of clinical samples are available, but the resemblances of tumorigenesis and autoimmune genesis in terms of immune responses remains to be summed up. METHODS: Considering the high disease prevalence, we chose invasive ductal carcinoma (IDC) and systemic lupus erythematosus (SLE) to study the potential commonalities of immune responses. We obtained gene expression data of IDC/SLE patients and normal controls from five IDC databases (GSE29044, GSE21422, GSE22840, GSE15852, and GSE9309) and five SLE databases (GSE154851, GSE99967, GSE61635, GSE50635, and GSE17755). We intended to identify genes differentially expressed in both IDC and SLE by using three bioinformatics tools including GEO2R, the limma R package, and Weighted Gene Co-expression Network Analysis (WGCNA) to perform function enrichment, protein-protein network, and signaling pathway analyses. RESULTS: The mRNA levels of signal transducer and activator of transcription 1 (STAT1), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase like (OASL), and PML nuclear body scaffold (PML) were found to be differentially expressed in both IDC and SLE by using three different bioinformatics tools of GEO2R, the limma R package and WGCNA. From the combined databases in this study, the mRNA levels of STAT1 and OAS1 were increased in IDC while reduced in SLE. And the mRNA levels of OASL and PML were elevated in both IDC and SLE. Based on Kyoto Encyclopedia of Genes and Genomes pathway analysis and QIAGEN Ingenuity Pathway Analysis, both IDC and SLE were correlated with the changes of multiple components involved in the Interferon (IFN)-Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. CONCLUSION: The expression levels of STAT1 and OAS1 manifest the opposite expression tendency across cancer and autoimmune disease. They are components in the IFN-JAK-STAT signaling pathway related to both tumorigenesis and autoimmune genesis. STAT1 and OAS1-associated IFN-JAK-STAT signaling could explain the commonalities during tumorigenesis and autoimmune genesis and render significant information for more precise treatment from the point of immune homeostasis.

Reshuffling of the ancestral core-eudicot genome shaped chromatin topology and epigenetic modification in Panax.

All extant core-eudicot plants share a common ancestral genome that has experienced cyclic polyploidizations and (re)diploidizations. Reshuffling of the ancestral core-eudicot genome generates abundant genomic diversity, but the role of this diversity in shaping the hierarchical genome architecture, such as chromatin topology and gene expression, remains poorly understood. Here, we assemble chromosome-level genomes of one diploid and three tetraploid Panax species and conduct in-depth comparative genomic and epigenomic analyses. We show that chromosomal interactions within each duplicated ancestral chromosome largely maintain in extant Panax species, albeit experiencing ca. 100-150 million years of evolution from a shared ancestor. Biased genetic fractionation and epigenetic regulation divergence during polyploidization/(re)diploidization processes generate remarkable biochemical diversity of secondary metabolites in the Panax genus. Our study provides a paleo-polyploidization perspective of how reshuffling of the ancestral core-eudicot genome leads to a highly dynamic genome and to the metabolic diversification of extant eudicot plants.

Exploring the Distinct Binding and Activation Mechanisms for Different CagA Oncoproteins and SHP2 by Molecular Dynamics Simulations.

CagA is a major virulence factor of Helicobacter pylori. H. pylori CagA is geographically subclassified into East Asian CagA and Western CagA, which are characterized by the presence of a EPIYA-D or EPIYA-C segment. The East Asian CagA is more closely associated with gastric cancer than the Western CagA. In this study, molecular dynamic (MD) simulations were performed to investigate the binding details of SHP2 and EPIYA segments, and to explore the allosteric regulation mechanism of SHP2. Our results show that the EPIYA-D has a stronger binding affinity to the N-SH2 domain of SHP2 than EPIYA-C. In addition, a single EPIYA-D binding to N-SH2 domain of SHP2 can cause a deflection of the key helix B, and the deflected helix B could squeeze the N-SH2 and PTP domains to break the autoinhibition pocket of SHP2. However, a single EPIYA-C binding to the N-SH2 domain of SHP2 cannot break the autoinhibition of SHP2 because the secondary structure of the key helix B is destroyed. However, the tandem EPIYA-C not only increases its binding affinity to SHP2, but also does not significantly break the secondary structure of the key helix B. Our study can help us better understand the mechanism of gastric cancer caused by Helicobacter pylori infection.

Exploring the Allosteric Mechanism of Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase 2 (SHP2) by Molecular Dynamics Simulations.

The Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2, encoded by PTPN11) is a critical allosteric phosphatase for many signaling pathways. Programmed cell death 1 (PD-1) could be phosphorylated at its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and can bind to SHP2 to initiate T cell inactivation. Although the interaction of SHP2-PD-1 plays an important role in the immune process, the complex structure and the allosteric regulation mechanism remain unknown. In this study, molecular dynamics (MD) simulations were performed to study the binding details of SHP2 and PD-1, and explore the allosteric regulation mechanism of SHP2. The results show that ITIM has a preference to bind to the N-SH2 domain and ITSM has almost the same binding affinity to the N-SH2 and C-SH2 domain. Only when ITIM binds to the N-SH2 domain and ITSM binds to the C-SH2 domain can the full activation of SHP2 be obtained. The binding of ITIM and ITSM could change the motion mode of SHP2 and switch it to the activated state.

Corrigendum: Dendrobium officinale Orchid Extract Prevents Ovariectomy-Induced Osteoporosis In Vivo and Inhibits RANKL-Induced Osteoclast Differentiation In Vitro.

[This corrects the article DOI: 10.3389/fphar.2017.00966.].

Current status and future prospects of stem cell therapy in Alzheimer's disease.

Alzheimer's disease is a common progressive neurodegenerative disorder, pathologically characterized by the presence of ß-amyloid plaques and neurofibrillary tangles. Current treatment approaches using drugs only alleviate the symptoms without curing the disease, which is a serious issue and influences the quality of life of the patients and their caregivers. In recent years, stem cell technology has provided new insights into the treatment of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Currently, the main sources of stem cells include neural stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. In this review, we discuss the pathophysiology and general treatment of Alzheimer's disease, and the current state of stem cell transplantation in the treatment of Alzheimer's disease. We also assess future challenges in the clinical application and drug development of stem cell transplantation as a treatment for Alzheimer's disease.

Natural borneol sensitizes human glioma cells to cisplatin-induced apoptosis by triggering ROS-mediated oxidative damage and regulation of MAPKs and PI3K/AKT pathway.

Context: Cisplatin-based chemotherapy was widely used in treating human malignancies. However, side effects and chemoresistance remains the major obstacle.Objective: To verify whether natural borneol (NB) can enhance cisplatin-induced glioma cell apoptosis and explore the mechanism.Materials and methods: Cytotoxicity of cisplatin and/or NB towards U251 and U87 cells were determined with the MTT assay. Cells were treated with 0.25-80 µg/mL cisplatin and/or 5-80 µM NB for 48 h. The effects of NB and/or cisplatin on apoptosis and cell cycle distribution were quantified by flow cytometric analysis. Protein expression was detected by western blotting. ROS generation was conducted by measuring and visualising an oxidation-sensitive fluorescein DCFH-DA.Results: NB synergistically enhanced the anticancer efficacy of cisplatin in human glioma cells. Co-treatment of 40 µg/mL NB and 40 µg/mL cisplatin significantly inhibited U251 cell viability from 100% to 28.2% and increased the sub-G1 population from 1.4% to 59.3%. Further detection revealed that NB enhanced cisplatin-induced apoptosis by activating caspases and triggering reactive oxygen species (ROS) overproduction as evidenced by the enhancement of green fluorescence intensity from 265% to 645%. ROS-mediated DNA damage was observed as reflected by the activation of ATM/ATR, p53 and histone. Moreover, MAPKs and PI3K/AKT pathways also contributed to co-treatment-induced U251 cell growth inhibition. ROS inhibition by antioxidants effectively improved MAPKs and PI3K/AKT functions and cell viability, indicating that NB enhanced cisplatin-induced cell growth in a ROS-dependent manner.Discussion and conclusions: Natural borneol had the potential to sensitise human glioma cells to cisplatin-induced apoptosis with potential application in the clinic.

Exploring the allosteric mechanism of protein tyrosine phosphatase 1B by molecular dynamics simulations.

Communicated by Ramaswamy H. Sarma.

Co-targeting CK2α and YBX1 suppresses tumor progression by coordinated inhibition of the PI3K/AKT signaling pathway.

Protein kinase CK2 alpha (CK2α) is involved in the development of multiple malignancies. Overexpression of Y-box binding protein 1 (YBX1) is related to tumor proliferation, drug resistance, and poor prognosis. Studies have demonstrated that both CK2 and YBX1 could regulate the PI3K/AKT pathway. In addition, we predicted that CK2 might be the upstream kinase of YBX1 through the Human Protein Reference Database (HPRD). Herein, we hypothesize that CK2 may interact with YBX1 and they regulate the PI3K/AKT signaling pathway together. Expressions of CK2α and YBX1 in cancer cell lines were evaluated by immunoblotting. The results showed that CK2α could regulate the expression of YBX1 at the transcriptional level, which is dependent on its enzymatic activity. Synergistic effects of PI3K/AKT pathway inactivation could be observed through combined inhibition of CK2α and YBX1, and YBX1 was required for CK2α-induced PI3K/AKT pathway activation. Further results demonstrated that CK2α could interact with YBX1 and PI3K/AKT antagonist decreased cell resistance to doxorubicin induced by co-activation of CK2α and YBX1. These results indicated that combined inhibition of CK2α and YBX1 showed synergistic effects in inactivating the PI3K/AKT signaling pathway and may be one of the mechanisms involved in tumor growth and migration.

Baicalin Regulates Proliferation, Apoptosis, Migration, and Invasion in Mesothelioma.

BACKGROUND Baicalin, one of the main bioactive components extracted from the traditional Chinese medicine baical Skullcap root, has an anti-tumor activity which had been studied in several cancers. However, its role in human mesothelioma remains unknown. In this study, we investigated the anti-tumor mechanisms of baicalin in the mesothelioma cell line MESO924. MATERIAL AND METHODS Effects of baicalin on mesothelioma were assessed by measuring cell viability, apoptosis, migration, invasion, inactivation of signaling intermediates, and cell-cycle alterations. RESULTS Baicalin inhibited the proliferation, migration, and invasion of human mesothelioma cells and increased their apoptosis, all in a dose-dependent manner. Specifically, baicalin decreased the expression of p-EGFR, p-AKT, p-MAPK, p-S6, Bcl-2, and VEGF and increased the expression of Bax in mesothelioma cells. The suppressed mesothelioma cellular proliferation is due to the arrest of the S cell cycle by baicalin. Inhibition of the PI3K/AKT/mTOR signaling pathway by a PI3K/AKT/mTOR inhibitor augmented the anti-proliferation effects induced by baicalin. In addition, baicalin increased the sensitivity of MESO924 to the chemotherapeutic drugs doxorubicin, cisplatin, and pemetrexed. CONCLUSIONS These results highlight the roles of baicalin in inhibiting cell growth, migration, and invasion of mesothelioma cells while increasing apoptosis and sensitizing cells to chemotherapeutic agents through the PI3K/AKT/mTOR signaling pathway, which indicates that baicalin could be a useful drug for mesothelioma therapy.

Enhanced anticancer efficiency of doxorubicin against human glioma by natural borneol through triggering ROS-mediated signal.

Doxorubicin (DOX) as a first-line chemotherapeutic drug has been widely used for therapy of human cancers. However, side effects and chemo-resistance severely blocked its clinic application. Herein, natural borneol (NB) as a novel monoterpenoid chemosensitizer was found to have the potential to increase the blood brain barrier (BBB) permeability and intracellular uptake of DOX in vitro, and synergistically enhanced DOX-induced cytotoxicity in human glioma cells. NB treatment significantly potentiated DOX-induced G2/M cell cycle arrest by triggering reactive oxygen species (ROS)-mediated DNA damage. NB also enhanced DOX-induced dysfunction of MAPKs and PI3 K/AKT pathways. Furthermore, U251 human glioma xenograft growth in vivo was also effectively inhibited by combined treatment of DOX with NB through induction of G2/M-phase arrest and antiangiogenesis. Taken together, our finding validated that NB could act as novel chemosensitizer to enhance DOX-induced anticancer efficacy, and strategy of using NB and DOX could be a high efficient way in therapy of human cancers.

Hypoglycemic effect and mechanism of isoquercitrin as an inhibitor of dipeptidyl peptidase-4 in type 2 diabetic mice.

Glucagon-like peptide (GLP)-1 is a potent glucose-dependent insulinotropic gut hormone released from intestinal L cells. The aim of this study was to investigate isoquercitrin as an inhibitor of dipeptidyl peptidase IV (DPP-IV) and determine whether it affects GLP-1 release in normal mice and NCI-H716 cells. In vitro, we used chromogenic substrate method detection methods to measure DPP-IV. We found that isoquercitrin was a competitive inhibitor, with IC50 and K i values of 96.8 and 236 µM, respectively. Isoquercitrin and sitagliptin also stimulated GLP-1 release in NCI-H716 cells. In vivo, a type 2 diabetic mouse model was established, and oral treatment with different concentration of isoquercitrin and sitagliptin for 8 weeks significantly decreased the fasting blood glucose level. The weight and the levels of serum GLP-1 and insulin of the mice in the isoquercitrin group were higher than those in the model group (P < 0.001). An oral glucose tolerance test showed that the isoquercitrin significantly inhibited postprandial blood glucose changes in a dose-dependent manner. These findings demonstrated the hypoglycemic effects of isoquercitrin and indicated that isoquercitrin improved insulin sensitivity by targeting DPP-IV.

Dendrobium officinale Orchid Extract Prevents Ovariectomy-Induced Osteoporosis in Vivo and Inhibits RANKL-Induced Osteoclast Differentiation in Vitro.

Background:Dendrobium officinale, a traditional Chinese medical herb with high value that is widely used in Asia, possesses many positive effects on human health, including anti-chronic inflammation, anti-obesity, and immune modulation properties; however, whether D. officinale has inhibitory effects on postmenopausal osteoporosis remains unknown. Objective: We investigated the effects of D. officinale extract (DOE) on ovariectomy-induced bone loss in vivo and on osteoclastogenesis in vitro. Methods:In vivo, female rats were divided into a sham-operated (sham) group and five ovariectomized (OVX) subgroups: OVX with vehicle (OVX), OVX with Xian-Ling-Gu-Bao capsule (240 mg/kg body weight/day), and OVX with low-, medium-, and high-dose DOE (150, 300, and 600 mg/kg body weight/day, respectively). Animals in each group were administered their corresponding treatments for 13 weeks. Body weight, serum biochemical parameters, uterine and femoral physical parameters, bone mineral density (BMD), bone biomechanical properties, and bone microarchitecture were obtained. In vitro, the effects of DOE on osteoclastogenesis were examined using RAW264.7 cells. The effects of DOE on osteoclastogenesis and the expression of osteoclast-specific marker genes and proteins were determined. Results: DOE effectively ameliorated serum biochemical parameters, especially alleviated estradiol (E2) deficiency and maintained calcium and phosphorus homeostasis. DOE improved uterine and femoral physical parameters. In addition, DOE improved femoral BMD and biomechanical properties. DOE significantly ameliorated bone microarchitecture. Moreover, DOE inhibited osteoclastogenesis independent of its cytoxicity and suppressed the expression of osteoclast-specific marker genes and proteins. Conclusion: DOE can effectively prevent ovariectomy-induced bone loss in vivo and inhibit osteoclastogenesis in vitro.

Identification of key genes associated with the effect of estrogen on ovarian cancer using microarray analysis.

PURPOSE: To identify key genes related to the effect of estrogen on ovarian cancer. METHODS: Microarray data (GSE22600) were downloaded from Gene Expression Omnibus. Eight estrogen and seven placebo treatment samples were obtained using a 2 × 2 factorial designs, which contained 2 cell lines (PEO4 and 2008) and 2 treatments (estrogen and placebo). Differentially expressed genes were identified by Bayesian methods, and the genes with P < 0.05 and |log2FC (fold change)| ≥0.5 were chosen as cut-off criterion. Differentially co-expressed genes (DCGs) and differentially regulated genes (DRGs) were, respectively, identified by DCe function and DRsort function in DCGL package. Topological structure analysis was performed on the important transcriptional factors (TFs) and genes in transcriptional regulatory network using tYNA. Functional enrichment analysis was, respectively, performed for DEGs and the important genes using Gene Ontology and KEGG databases. RESULTS: In total, 465 DEGs were identified. Functional enrichment analysis of DEGs indicated that ACVR2B, LTBP1, BMP7 and MYC involved in TGF-beta signaling pathway. The 2285 DCG pairs and 357 DRGs were identified. Topological structure analysis showed that 52 important TFs and 65 important genes were identified. Functional enrichment analysis of the important genes showed that TP53 and MLH1 participated in DNA damage response and the genes (ACVR2B, LTBP1, BMP7 and MYC) involved in TGF-beta signaling pathway. CONCLUSION: TP53, MLH1, ACVR2B, LTBP1 and BMP7 might participate in the pathogenesis of ovarian cancer.

Epigallocatechin-3-gallate (EGCG) inhibits 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of glycerol.

The inhibition of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) is considered able to decrease serum cholesterol levels and dramatically reduce the risk for cardiovascular and cerebrovascular diseases. The statins, competitive inhibitors of HMGCR, have been employed to control hypercholesterolemia. But their side effects, especially their safety of long-term administration have attracted great attention. Therefore, there is still an urgent requirement for the development of safer inhibitors of HMGCR with less serious side effects. In this study, we cloned and purified the catalytic domain of human HMGCR (△HMGCR), and applied the method of Ultra Performance Liquid Chromatography (UPLC) to assay △HMGCR activity and screen its inhibitors from natural products. The results indicated that EGCG can inhibit △HMGCR in the presence of some glycerol in vitro and can decrease cellular total cholesterol in HepG2 cells. As a consequence, it is promising to put EGCG into the development of hypolipidemic health product.

Caenorhabditis elegans eyes absent ortholog EYA-1 is required for stress resistance.

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that regulates multiple developmental processes throughout the metazoans. It is a dual function protein, working as a transcription factor in the nucleus and as a tyrosine phosphatase in the cytoplasm. In this study, we isolated EYA-1 of Caenorhabditis elegans, the only homolog of Eyes absent, and set up an effective feeding-based RNAi (RNA interference) against the gene. We found that knockdown of EYA-1 decreased heat and oxidative stress tolerance and accelerated the onset of paralysis mediated by Aß1-42 proteotoxicity and polyQ. Under heat stress (35°C), EYA-1 knockdown shortened the mean lifespan by 16.8%, which could be attributed to decrease in heat shock protein-16.2 (hsp-16.2) expression. Under oxidative stress, EYA-1 knockdown could shorten the mean lifespan by 18.7%, which could be attributed to intracellular ROS accumulation and the decrease of superoxide dismutase-3 (sod-3) protein expression. Moreover, EYA-1 knockdown animals also showed increased lipofuscin accumulation under oxidative stress. Further studies demonstrated that EYA-1 knockdown could not inhibit daf-16 nuclear accumulation in wild-type worms in response to stress. On the other hand, EYA-1 deficiency did not further reduce stress resistance of daf-16 mutants, which are stress sensitive. Quantitative real-time PCR results also showed that the expression of two daf-16 target genes, hsp-12.3 and sod-3, was downregulated in EYA-1 RNAi-treated worms under stress. All this evidence indicates EYA-1 is required for stress resistance of worms, and it might act downstream of daf-16 to regulate expression of stress resistance-associated genes.

Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity.

AIM: To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. METHODS: Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K(+) efflux and Na(+) influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. RESULTS: Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 µg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K(+) efflux and Na(+) influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. CONCLUSION: Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections.

Icaritin causes sustained ERK1/2 activation and induces apoptosis in human endometrial cancer cells.

Icaritin, a compound from Epimedium Genus, has selective estrogen receptor (ER) modulating activities, and possess anti-tumor activity. Here, we examined icaritin effect on cell growth of human endometrial cancer Hec1A cells and found that icaritin potently inhibited proliferation of Hec1A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinD1 and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase1/2 (the MAPK/ ERK1/2) in Hec1A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. Our results demonstrated that icaritin induced sustained ERK 1/2 activation and inhibited growth of endometrial cancer Hec1A cells, and provided a rational for preclinical and clinical evaluation of icaritin for endometrial cancer therapy.

ER-α36, a novel variant of ER-α, mediates estrogen-stimulated proliferation of endometrial carcinoma cells via the PKCδ/ERK pathway.

BACKGROUND: Recently, a variant of ER-α, ER-α36 was identified and cloned. ER-α36 lacks intrinsic transcription activity and mainly mediates non-genomic estrogen signaling. The purpose of this study was to investigate the function and the underlying mechanisms of ER-α36 in growth regulation of endometrial Ishikawa cancer cells. METHODS: The cellular localization of ER-α36 and ER-α66 were determined by immunofluorescence in the Ishikawa cells. Ishikawa endometrial cancer control cells transfected with an empty expression vector, Ishikawa cells with shRNA knockdown of ER-α36 (Ishikawa/RNAiER36) and Ishikawa cells with shRNA knockdown of ER-α66 (Ishikawa/RNAiER66) were treated with E2 and E2-conjugated to bovine serum albumin (E2-BSA, membrane impermeable) in the absence and presence of different kinase inhibitors HBDDE, bisindolylmaleimide, rottlerin, H89 and U0126. The phosphorylation levels of signaling molecules and cyclin D1/cdk4 expression were examined with Western blot analysis and cell growth was monitored with the MTT assay. RESULTS: Immunofluorescence staining of Ishikawa cells demonstrated that ER-α36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-α66 was predominantly localized in the cell nucleus. Both E2 and E2-BSA rapidly activated PKCδ not PKCα in Ishikawa cells, which could be abrogated by ER-α36 shRNA expression. E2-and E2-BSA-induced ERK phosphorylation required ER-α36 and PKCδ. However, only E2 was able to induce Camp-dependent protein kinase A (PKA) phosphorylation. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-α36. CONCLUSION: E2 activates the PKCδ/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-α36, suggesting a possible involvement of ER-α36 in E2-dependent growth-promoting effects in endometrial cancer cells.