Upregulation of glutamate transporter 1 by mTOR/Akt pathway in astrocyte culture during oxygen-glucose deprivation and reoxygenation.

Astrocyte-specific glutamate transporter subtype 1 (GLT-1) plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons. Although the mammalian target of rapamycin (mTOR)/protein kinase B(Akt)/nuclear factor kappa B signaling cascade is involved in the upregulation of astrocytic GLT-1 in oxygen-glucose deprivation (OGD), it is unclear whether the mTOR/Akt pathway is involved in astrocytic GLT-1 upregulation in OGD and reoxygenation (OGD/R). In this study, we found that the treatment of cultured astrocytes with rapamycin and triciribine led to the decreased astrocytes' protrusions, smaller nuclei, and an increased apoptotic rate. The inhibitors of mTOR complex 1 significantly increased the expression levels of phosphorylated Akt-Ser473 (p-Akt), phosphorylated Akt-Thr308(p-Akt), and GLT-1, while Akt-specific inhibitors blocked GLT-1 expression, suggesting that the mTOR/Akt pathway is involved in GLT-1 upregulation. We further demonstrated that astrocytes under OGD/R adapted to environmental changes through the mTOR/Akt pathway, mainly by altering cell morphology and apoptosis and upregulating the expression levels of p-Akt and GLT-1. Our results suggested that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating cell morphology, apoptosis and GLT-1 upregulation.

Construction and validation of a m6A RNA methylation and ferroptosis-related prognostic model for pancreatic cancer by integrated bioinformatics analysis.

Background: Both N6-methyladenosine (m6A) ribonucleic acid (RNA) methylation and ferroptosis regulators are demonstrated to have significant effects on the malignant clinicopathological characteristics of pancreatic adenocarcinoma (PAAD) patients. However, the currently available clinical indexes are not sufficient to predict precise prognostic outcomes pf PAAD patients accurately. This study aims to examine the clinicopathologic features of m6A RNA methylation and ferroptosis regulators in predicting the outcomes of different types of cancer. Methods: As the foundation for this research, the differentially expressed genes (DEGs) between PAAD tissues and adjacent normal tissues were first identified. Next, dimensional reduction analysis (DCA) based on m6A RNA methylation regulators and ferroptosis regulators were performed and DEGs between good/poor prognosis PAAD patient clusters were identified. DEGs were then screened by Cox analysis, and finally a risk signature was established by least absolute shrinkage and selection operator (LASSO) analyses. The prediction model based on risk score was further evaluated by a validation set from Gene Expression Omnibus (GEO) database. Results: In total, 4 m6A RNA methylation regulator genes and 29 ferroptosis regulator genes were found to have close causal relationships with the prognosis of PAAD, and a risk score with 3 m6A methylation regulators (i.e., IGF2BP2, IGF2BP3, and METTL16) and 4 ferroptosis regulators (i.e., ENPP2, ATP6V1G2, ITGB4, and PROM2) was constructed and showed to be highly involved in PAAD progression and could serve as effective markers for prognosis with AUC value equaled 0.753 in training set and 0.803 in validation set. Conclusions: The combined prediction model, composed of seven regulators of m6A methylation and ferroptosis, in this study more effectively reflects the progression and prognosis of PAAD than previous single genome or epigenetic analysis. Our study provides a broader perspective for the subsequent establishment of prognostic models and the patients may benefit from more precision management.

Comparison of full-field optical coherence tomography imaging for pancreatic tissue sample obtained by EUS-fine-needle biopsy and conventional histological examination: A study protocol for a prospective trial.

For a definitive diagnosis of fine-needle aspiration (FNA)/biopsy, one of the reliable techniques to determine the adequacy and accuracy rapid on-site evaluation (ROSE) of cytological samples is preferable. Because of the lack of trained pathologists, alternatives have to be explored. This study is primarily conducted to determine the diagnostic sensitivity and specificity of full-field optical coherence tomography (FF-OCT) and secondarily to evaluate the possibility of FF-OCT differentiating different types of pancreatic diseases. The diagnostic coherence of FF-OCT by a trained assistant (endoscopist) and trained pathologist is also compared. This is a single-center, prospective, observation trial. Eighty patients would be enrolled in the study. The tissue samples acquired by endoscopic ultrasound fine-needle biopsy (EUS-FNB) would be imaged by the FF-OCT system, interpreted by a trained endoscopist and a pathologist. The results of the image interpretation would be verified with histological findings. This study determines the diagnostic capability of FF-OCT as a ROSE technique while performing EUS-FNB, and whether endoscopists can implement the assessment.

Correlation between S100A11 and the TGF-ß1/SMAD4 pathway and its effects on the proliferation and apoptosis of pancreatic cancer cell line PANC-1.

S100A11 as a S100 protein family member has been documented to play dual-direction regulation over cancer cell proliferation. We explored the role of S100A11 in the proliferation and apoptosis of pancreatic cancer cell line PANC-1 and the potential mechanisms involving the TGF-ß1/SMAD4/p21 pathway. S100A11 and TGF-ß1 protein expressions in 30 paraffin-embedded specimens were evaluated by immunohistochemistry. S100A11 and TGF-ß1 expression in PANC-1 cell line was suppressed using small interfering RNA (siRNA), respectively. Subsequently, pancreatic cancer cell apoptosis was measured by Cell Counting Kit-8 and flow cytometry, and S100A11 and TGF-ß1/SMAD4/p21 pathway proteins and genes were detected with Western blotting and quantitative polymerase chain reaction (qPCR). S100A11 cytoplasmic/nuclear protein translocation was examined using NE-PER® cytoplasm/nuclear protein extraction in cells interfered with TGF-ß1 siRNA. Our results showed that S100A11 expression was positively correlated with TGF-ß1 expression in pancreatic cancerous tissue. Silencing TGF-ß1 down-regulated intracellular P21WAF1 expression by 90%, blocked S100A11 from cytoplasm entering nucleus, and enhanced cell proliferation. Silencing S100A11 down-regulated intracellular P21 expression and promoted cell apoptosis without significantly changing TGF-ß1 and SMAD4 expression. Our findings revealed that S100A11 and TGF-ß1/SMAD4 signaling pathway were related but mutually independent in regulating PANC-1 cells proliferation and apoptosis. Other independent mechanisms might be involved in S100A11's regulation of pancreatic cell growth. S100A11 could be a potential gene therapy target for pancreatic cancer.

Sex comb on midleg like-2 is a novel specific marker for the diagnosis of gastroenteropancreatic neuroendocrine tumors.

Sex comb on midleg like-2 (SCML2) is a polycomb-group protein that encodes transcriptional repressors essential for appropriate development in the fly and in mammals. On the basis of previous findings, the present study aimed to explore the possibility of developing SCML2 into a new diagnostic marker for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). A total of 64 paired GEP-NET tissues and adjacent non-tumorous tissues were obtained from patients who had undergone surgical resection between January 2009 and January 2014, and the expression of SCML2 and two neuroendocrine markers, namely synaptophysin (Syn) and chromogranin A (CgA), in the tissues was assessed by immunohistochemistry. Strong SCML2 staining was observed predominantly in the cell nuclei of GEP-NET tissues, and the overall expression rate and staining intensity of SCML2 were higher than those of Syn or CgA, respectively. Spearman rank correlation analysis demonstrated that SCML2 was not correlated with either Syn or CgA, while the combined detection of SCML2 with Syn or with CgA increased the diagnostic sensitivity to 100%. SCML2 expression in GEP-NETs was associated with several clinicopathological parameters, such as histological type, tumor grade, depth of invasion and clinical stage. Kaplan-Meier survival curves revealed that patients with higher SCML2 expression had lower survival rates than those with lower expression levels, while Cox proportional hazards regression analysis revealed that SCML2 was not an independent prognostic factor for GEP-NET patients. Therefore, SCML2 may have potential as a specific marker for joint use with other markers to improve the diagnostic efficiency of GEP-NETs.

S100 family signaling network and related proteins in pancreatic cancer (Review).

The occurrence and development of pancreatic cancer is a complex process convoluted by multi-pathogenies, multi-stages and multi-factors. S100 proteins are members of the S100 family that regulate multiple cellular pathways related to pancreatic cancer progression and metastasis. S100 proteins have a broad range of intracellular and extracellular functions, including the regulation of protein phosphorylation and enzyme activity, calcium homeostasis and the regulation of cytoskeletal components and transcriptional factors. S100 proteins interact with receptor for advanced glycation end-products (RAGE), p53 and p21, which play a role in the degradation of the extracellular matrix (ECM) and metastasis, and also interact with cytoskeletal proteins and the plasma membrane in pancreatic cancer progression and metastasis. S100A11 and S100P are significant tumor markers for pancreatic cancer and unfavorable predictors for the prognosis of patients who have undergone surgical resection. Recently, S100A2 has been suggested to be a negative prognostic biomarker in pancreatic cancer, and the expression of S100A6 may be an independent prognostic impact factor. The expression of S100A4 and S100P is associated with drug resistance, differentiation, metastasis and clinical outcome. This review summarizes the role and significance of the S100 family signaling network and related proteins in pancreatic cancer.

Upregulation of glutamate transporter GLT-1 by mTOR-Akt-NF-кB cascade in astrocytic oxygen-glucose deprivation.

Excessive extracellular glutamate leads to neuronal death in central nervous system. Excitatory glutamate transporter subtype 2 (GLT-1) carries bulk of glutamate reuptake in cerebral ischemia. Although GLT-1 expression fluctuates during the period of ischemia, little is known about its regulatory mechanism. Here we show an up-regulation of GLT-1 via mammalian target of rapamycin (mTOR)-Akt-nuclear factor-кB (NF-кB) signaling cascade in oxygen glucose deprivation (OGD). We found that brief rapamycin treatment significantly increased GLT-1 expression in cultured astrocytes. Rapamycin increased phosphorylation of raptor at Ser792 and decreased phosphorylation of rictor at Thr1135, suggesting that both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) are involved in GLT-1 expression. This conclusion was further confirmed by raptor and rictor disruption experiments. Akt was activated by mTORC1 inhibition and required for GLT-1 expression because triciribine, a specific inhibitor of Akt, blocked the increase of GLT-1 expression. mTOR-Akt cascade then activated NF-кB and increased кB-motif-binding phosphoprotein (KBBP) expression and GLT-1 transcription. We next demonstrated that mTOR-Akt-NF-кB cascade was activated in OGD and subsequently caused the upregulation of GLT-1. Supporting evidence included: (1) inhibition of Akt or NF-кB occluded OGD-induced GLT-1 upregulation; (2) Raptor knock-down plus OGD did not add to the increase of GLT-1 expression; (3) Intact mTORC2 was required for GLT-1 enhancement. In summary, our data first showed that mTOR-Akt-NF-кB cascade played critical roles to up-regulate GLT-1 in OGD. This signaling cascade may work to promote glutamate uptake in brain ischemia and neurodegenerative diseases.

Insulin increases glutamate transporter GLT1 in cultured astrocytes.

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.