Multimodal Imaging of Orthotopic Mouse Model of Endometrial Carcinoma

Background

Orthotopic endometrial cancer models provide a unique tool for studies of tumour growth and metastatic spread. Novel preclinical imaging methods also have the potential to quantify functional tumour characteristics in vivo, with potential relevance for monitoring response to therapy.

Methods

After orthotopic injection with luc-expressing endometrial cancer cells, eleven mice developed disease detected by weekly bioluminescence imaging (BLI). In parallel the same mice underwent positron emission tomography–computed tomography (PET-CT) and magnetic resonance imaging (MRI) employing ¹⁸F-fluorodeoxyglocose (¹⁸F-FDG) or ¹⁸F- fluorothymidine (¹⁸F-FLT) and contrast reagent, respectively. The mice were sacrificed when moribund, and post-mortem examination included macroscopic and microscopic examination for validation of growth of primary uterine tumours and metastases. PET-CT was also performed on a patient derived model (PDX) generated from a patient with grade 3 endometrioid endometrial cancer.

Results

Increased BLI signal during tumour growth was accompanied by increasing metabolic tumour volume (MTV) and increasing MTV x mean standard uptake value of the tumour (SUV_mean) in ¹⁸F-FDG and ¹⁸F-FLT PET-CT, and MRI conspicuously depicted the uterine tumour. At necropsy 82% (9/11) of the mice developed metastases detected by the applied imaging methods. ¹⁸F-FDG PET proved to be a good imaging method for detection of patient derived tumour tissue.

Conclusions

We demonstrate that all imaging modalities enable monitoring of tumour growth and metastatic spread in an orthotopic mouse model of endometrial carcinoma. Both PET tracers, ¹⁸F-FDG and ¹⁸F-FLT, appear to be equally feasible for detecting tumour development and represent, together with MRI, promising imaging tools for monitoring of patient-derived xenograft (PDX) cancer models.     

自噬在声动力疗法抑制C6胶质瘤细胞增殖中的作用

目的:探讨自噬在血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(Sonodynamic therapy,SDT)抑制C6胶质瘤细胞增殖中的作用。方法:选取对数期生长的C6胶质瘤细胞并随机分为四组:对照组(未予处理)、超声组(单独超声照射)、HMME组(单独加入HMME)、SDT组(超声照射+HMME)。透射电镜观察SDT处理的C6胶质瘤细胞中自噬体数量的改变。应用qRT-PCR和免疫印迹分析SDT处理对C6胶质瘤细胞中的LC3、Beclin1、Bcl-2 m RNA及蛋白表达水平的影响。MTT检测C6胶质瘤细胞的活力变化。结果:透射电子显微镜显示SDT组自噬体数量较对照组明显增多。SDT组C6胶质瘤细胞中微管相关蛋白1轻链3 (Microtubule associated protein 1 light chain 3, LC3)、Beclin1 m RNA和蛋白水平高于对照组,B细胞淋巴瘤-2(B cell lymphoma-2, Bcl-2) m RNA和蛋白水平低于对照组。与对照组相比,SDT组C6胶质瘤细胞存活率从0 h至6 h逐渐下降,从12 h至72 h逐渐升高。3-甲基腺嘌呤(3-Methyladenine,3-MA)+SDT、氯喹(Chloroquine,CQ)+SDT处理后C6胶质瘤细胞存活率较SDT组明显降低。结论:SDT可能通过诱导自噬抑制C6胶质瘤细胞增殖。     

Establishment of an Animal Model of Bisphosphonate-Related Osteonecrosis of the Jaws in Spontaneously Diabetic Torii Rats

Background

We evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ).

Materials and Methods

Male Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies.

Results

In the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group.

Conclusions

This study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ.     

Role of Sarcoplasmic Reticulum Calcium in Development of Secondary Calcium Rise and Early Afterdepolarizations in Long QT Syndrome Rabbit Model

Background

L-type calcium current reactivation plays an important role in development of early afterdepolarizations (EADs) and torsades de pointes (TdP). Secondary intracellular calcium (Ca_i) rise is associated with initiation of EADs.

Objective

To test whether inhibition of sarcoplasmic reticulum (SR) Ca²⁺ cycling suppresses secondary Ca_i rise and genesis of EADs.

Methods

Langendorff perfusion and dual voltage and Ca_i optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB) was created by radiofrequency ablation. After baseline studies, E4031, SR Ca²⁺ cycling inhibitors (ryanodine plus thapsigargin) and nifedipine were then administrated subsequently, and the protocols were repeated.

Results

At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD) was significantly prolonged and the amplitude of secondary Ca_i rise was enhanced, and 7 (70%) rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced (2 hearts, 20%, p = 0.03). Although APD was significantly prolonged (from 298 ± 30 ms to 457 ± 75 ms at pacing cycle length of 1000 m, p = 0.007) by ryanodine plus thapsigargin, the secondary Ca_i rise was suppressed (from 8.8 ± 2.6% to 1.2 ± 0.9%, p = 0.02). Nifedipine inhibited TdP inducibility in all rabbit hearts.

Conclusion

In this AVB and long QT rabbit model, inhibition of SR Ca²⁺ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Ca_i rise and genesis of EADs.     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

Circulating Adiponectin and Risk of Endometrial Cancer

Background

Adiponectin is an insulin-sensitizing hormone produced by adipocytes. It has been suggested to be involved in endometrial tumorigenesis. Published data have shown inconsistent results for the association between circulating adiponectin levels and endometrial cancer. In this study, we conducted a meta-analysis to evaluate the predictive value of circulating adiponectin levels on the development of endometrial cancer.

Methods

PubMed, Embase, ISI web of knowledge, and Cochrane databases were searched for all eligible studies, and the summary relative risk (SRR) was calculated. Additionally, we performed dose-response analysis with eight eligible studies.

Results

A total of 1,955 cases and 3,458 controls from 12 studies were included. The SRR for the ‘highest’ vs ‘lowest’ adiponectin levels indicated high adiponectin level reduced the risk of endometrial cancer [SRR = 0.40, 95% confidence interval (CI), 0.33–0.66]. Results from the subgroup analyses were consistent with the overall analysis. The SRR for each 1 µg/ml increase of adiponectin indicated a 3% reduction in endometrial cancer risk (95% CI: 2%–4%), and a 14% reduction for each increase of 5 µg/ml (95% CI: 9%–19%). No evidence of publication bias was found.

Conclusions

This meta-analysis demonstrates that low level of circulating adiponectin is a risk factor for endometrial cancer.     

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Objective

Computational models of calcium (Ca²⁺) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²⁺ signaling model for RPE based on our experimental data of mechanically induced Ca²⁺ wave in the in vitro model of RPE, the ARPE-19 monolayer.

Methods

We combined six essential Ca²⁺ signaling components into a model: stretch-sensitive Ca²⁺ channels (SSCCs), P₂Y₂ receptors, IP₃ receptors, ryanodine receptors, Ca²⁺ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²⁺ waves reproduced our control experimental data and data where gap junctions were blocked.

Results

Our model was able to explain Ca²⁺ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²⁺ through plasma membrane SSCCs and gap junctions conduct the Ca²⁺ and IP³ between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²⁺ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP₃ receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca²⁺ signal in the distance.

Conclusions

The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P₂Y₂ receptors or accelerate P₂Y₂ receptor phosphorylation, which may partially be the reason for Ca²⁺ wave attenuation by suramin. Being the first RPE Ca²⁺ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.     

Triage of Atypical Glandular Cell by SOX1 and POU4F3 Methylation: A Taiwanese Gynecologic Oncology Group (TGOG) Study

Introduction

Invasive procedures including loop electrosurgical excision, cervical conization, and endometrial sampling are often recommended when atypical glandular cells (AGC) are detected on Pap smear with unsatisfactory colposcopy. These invasive procedures may result in patient anxiety, increased medical expense, and increasing the risk of preterm delivery in subsequent pregnancies. This study was performed to assess methylation biomarkers in the triage of AGC on Pap smear for invasive procedures.

Methods

We conducted a multicenter study in 13 medical centers in Taiwan from May 2012 to May 2014. A total of 55 samples diagnosed “AGC not otherwise specified” (AGC-NOS) were included. All patients with AGC underwent colposcopy, cervical biopsy, endometrial sampling, and conization if indicated. Multiplex quantitative methylation-specific polymerase chain reaction (QMSPCR) was performed. Sensitivity, specificity, and accuracy were calculated for detecting CIN3⁺ and endometrial complex hyperplasia.

Results

In 55 patients with AGC, the sensitivity for methylated (^m) SOX1^m, PAX1 ^m, ZNF582^m,PTPRR^m, AJAP1^m, HS3ST2^m, and POU4F3^m for detecting CIN3⁺ and endometrial complex hyperplasia lesions was 100, 86, 71, 86, 86, 57, and 100%; specificity was 67, 79, 85, 50, 52, 96, and 52%, respectively. Testing for high risk-HPV had a sensitivity of 57% and specificity of 75% for CIN3+ and endometrial complex hyperplasia lesions.

Conclusion

Methylated (^m) SOX1^m and POU4F3^m could be new methylation biomarkers for detection of CIN3⁺ and endometrial complex hyperplasia in AGC. Women with AGC and positive SOX1^m / POU4F3^m, colposcopy, cervical conization or endometrial sampling should be considered.     

Cancer-Testis Antigen Expression in Serous Endometrial Cancer with Loss of X Chromosome Inactivation

Background

Cancer-testis antigens (CTAs) are potential targets for cancer immunotherapy. Many CTAs are located on the X chromosome and are epigenetically regulated. Loss of X chromosome inactivation (XCI) is observed in breast and ovarian cancers and is thought to be related to the overexpression of CTAs. We investigated the relation between expression of CTAs and loss of XCI in endometrial cancer.

Materials and Methods

We used data generated by The Cancer Genome Atlas Genome Data Analysis Centers and data for Xist knockout mice available at the Gene Expression Omnibus.

Results

The status of XCI was estimated by methylation status, and deletion or gain of the X chromosome. The endometrial cancers were classified into the following three groups: preserved inactivated X chromosome (Xi) (n = 281), partial reactivation of Xi (n = 52), and two copies of active X group (n = 38). Loss of XCI was more common in serous adenocarcinoma. Expression of CTAs increased in endometrial cancer with loss of XCI, which was accompanied by global hypomethylation. Expression of CTAs did not increase in Xist knockout mice.

Conclusions

Loss of XCI is common in serous adenocarcinoma. Global hypomethylation, and not loss of XCI, is the main mechanism of overexpression of CTAs.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

Epithelial Cell Mitochondrial Dysfunction and PINK1 Are Induced by Transforming Growth Factor- Beta1 in Pulmonary Fibrosis

Background

Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis.

Methods

We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis.

Results

Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1 ^-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis.

Conclusion

TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.     

Ambient Fine Particulate Matter Suppresses In Vivo Proliferation of Bone Marrow Stem Cells through Reactive Oxygen Species Formation

Aims

Some environmental insults, such as fine particulate matter (PM) exposure, significantly impair the function of stem cells. However, it is unknown if PM exposure could affect the population of bone marrow stem cells (BMSCs). The present study was to investigate the effects of PM on BMSCs population and related mechanism(s).

Main Metheods

PM was intranasally distilled into male C57BL/6 mice for one month. Flow cytometry with antibodies for BMSCs, Annexin V and BrdU ware used to determine the number of BMSCs and the levels of their apoptosis and proliferation in vivo. Phosphorylated Akt (P-Akt) level was determined in the BM cells with western blotting. Intracellular reactive oxygen species (ROS) formation was quantified using flow cytometry analysis. To determine the role of PM-induced ROS in BMSCs population, proliferation, and apotosis, experiments were repeated using N-acetylcysteine (NAC)-treated wild type mice or a triple transgenic mouse line with overexpression of antioxidant network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase-1 with decreased in vivo ROS production.

Key Findings

PM treatment significantly reduced BMSCs population in association with increased ROS formation, decreased P-Akt level, and inhibition of proliferation of BMSCs without induction of apoptosis. NAC treatment or AON overexpression with reduced ROS formation effectively prevented PM-induced reduction of BMSCs population and proliferation with partial recovery of P-Akt level.

Significance

PM exposure significantly decreased the population of BMSCs due to diminished proliferation via ROS-mediated mechanism (could be partially via inhibition of Akt signaling).     

Dietary Inulin Fibers Prevent Proton-Pump Inhibitor (PPI)-Induced Hypocalcemia in Mice

Background

Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the most recognized side effect of proton-pump inhibitors (PPIs). Additionally, PPIH is associated with hypocalcemia and hypokalemia. It is hypothesized that PPIs reduce epithelial proton secretion and thereby increase the pH in the colon, which may explain the reduced absorption of and Mg²⁺ and Ca²⁺. Fermentation of dietary oligofructose-enriched inulin fibers by the microflora leads to acidification of the intestinal lumen and by this enhances mineral uptake. This study aimed, therefore, to improve mineral absorption by application of dietary inulin to counteract PPIH.

Methods

Here, C57BL/J6 mice were supplemented with omeprazole and/or inulin. Subsequently, Mg²⁺ and Ca²⁺ homeostasis was assessed by means of serum, urine and fecal electrolyte measurements. Moreover, the mRNA levels of magnesiotropic and calciotropic genes were examined in the large intestine and kidney by real-time PCR.

Results

Treatment with omeprazole significantly reduced serum Mg²⁺ and Ca²⁺ levels. However, concomitant addition of dietary inulin fibers normalized serum Ca²⁺ but not serum Mg²⁺ concentrations. Inulin abolished enhanced expression of Trpv6 and S100g in the colon by omeprazole. Additionally, intestinal and renal mRNA levels of the Trpm6 gene were reduced after inulin intake.

Conclusions

This study suggests that dietary inulin counteracts reduced intestinal Ca²⁺ absorption upon PPI treatment. In contrast, inulin did not increase intestinal absorption of Mg²⁺ sufficiently to recover serum Mg²⁺. The clinical potential of dietary inulin treatment should be the subject of future studies.     

Store-Operated Ca2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

Aims

Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and Results

We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca²⁺ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions

These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca²⁺ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.     

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

Purpose

Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated.

Methods

Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD⁺ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy.

Results

Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P < 0.01), 14% increase in ROS production (P < 0.01), and 81% reduction in mitochondrial membrane potential (P < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, P < 0.01) and increased the NADH/NAD⁺ ratio (46%, P < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation.

Conclusions

We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons.     

Multivariate Analysis of Prognostic Biomarkers in Surgically Treated Endometrial Cancer

Objective

The aim of this study was to identify biomarkers with prognostic value in the setting of surgically treated endometrial cancer.

Methods

Medical data for 282 patients with surgically treated endometrial cancer were reviewed retrospectively. Preoperative concentrations of six serum biomarkers (CA125, CA15-3, C-reactive protein [CRP], D-dimer [D-D], platelet-to-lymphocyte ratio [PLR], and neutrophil-to-lymphocyte ratio [NLR]) were analysed to determine potential associations with clinicopathologic characteristics and to assess prognostic values separately via Kaplan-Meier method and multivariate Cox regression.

Results

In univariate analyses, the 5-year overall survival (OS) rate was 86.5% for a maximum follow-up period of 75 months. High concentrations of CA125, CA15-3, CRP, D-D, PLR, and NLR each proved significantly predictive of poor survival (log-rank test, P<0.01). CRP and D-D were identified as independent prognosticators, using a Cox regression model. Study patients were then stratified (based on combined independent risk factors) into three tiers (P<0.001), marked by 5-year OS rates of 92.1%, 78.4%, and 33.3%.

Conclusions

All serum biomarkers assessed (CA125, CA15-3, CRP, D-D, PLR, and NLR) proved to be valid prognostic indices of surgically treated endometrial cancer. A novel prognostic grouping system, incorporating independent risk factors (CRP and D-D Concentrations), may have merit in assessing these patients preoperatively, providing a biologic basis for improved clinical staging.     

Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

Background

Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.

Methods

The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O₂ ⁻, H₂O₂, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.

Results

In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca²⁺, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca²⁺ into the cytoplasm after heat stress.

Conclusion

Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca²⁺, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.     

NOX4 Mediates BMP4-Induced Upregulation of TRPC1 and 6 Protein Expressions in Distal Pulmonary Arterial Smooth Muscle Cells

Rationale

Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.

Methods

We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca²⁺ determination in PASMCs.

Results

In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca²⁺]_i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H₂O₂ 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca²⁺]_i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.

Conclusion

These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.