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Jie Sun, Yan Liu, Jinhui Yu, Jin Wu, Wenting Gao, Liyuan Ran, Rujiao Jiang, Meihua Guo, Dongyu Han, Bo Liu, Ning Wang, Youwei Li, He Huang, Li Zeng, Ying Gao, Xin Li, and Yingjie Wu

Astragalus polysaccharide (APS) is the main component of Astragalus membranaceus, an anti-diabetic herb being used for thousands of years in Traditional Chinese medicine (TCM). In this study, we aimed to evaluate the impact of APS on hepatic insulin signaling, autophagy and ER stress response in high-fat-diet (HFD)-induced insulin resistance (IR) mice. APS was intra-gastrically administrated and metformin was used as a control medicine. Apart from monitoring the changes in the important parameters of IR progression, the gene and protein expression of the key factors marking the state of hepatic ER stress and autophagic flux were examined. We found that, largely comparable to the metformin regime, APS treatment resulted in an overall improvement of IR, as indicated by better control of body weight and blood glucose/lipid levels, recovery of liver functions and regained insulin sensitivity. In particular, the excessive and pro-apoptotic ER stress response and inhibition of autophagy, as a result of prolonged HFD exposure, were significantly corrected by APS administration, indicating a switch of the cellular fate in favor of cell survival. Using the HepG2/IR cell model, we demonstrated that APS modulated the insulin-initiated phosphorylation cascades in a similar manner to metformin. This study provides a rationale for exploiting the insulin-sensitizing potential of APS, which has a therapeutic performance almost equivalent to metformin, to enrich our options in the treatment of IR.

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Tae Woo Jung, Hyoung-Chun Kim, Yong Kyoo Shin, Hyeyoung Min, Seong-Wan Cho, Zi Soo Kim, Su Mi Han, A M Abd El-Aty, Ahmet Hacımüftüoğlu, and Ji Hoon Jeong

An aqueous extract of Humulus japonicus (AH) has been documented to ameliorate hypertension and non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effects of an aqueous extract of AH on thermogenesis and palmitate-induced oxidative stress in adipocytes. To verify the effect of AH on browning, we measured the expression levels of specific markers in 3T3-L1 adipocytes using qPCR and Western blotting, respectively. To assess the role of oxidative stress, cells were stained with DCFDA and observed by fluorescence microscopy. AH increased the expression of brown adipose tissue-specific markers. Additionally, it induced fatty acid oxidation and lipolysis and suppressed both lipogenic markers and lipid accumulation. Furthermore, AH ameliorated hydrogen peroxide-induced oxidative stress. Enhanced expression of these markers contributed to fat browning, fatty acid oxidation and lipolysis of 3T3-L1 adipocytes via the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor delta (PPARδ) signaling pathways. Moreover, AMPK and PPARδ resulting in protective effects of AH against oxidative stress. In sum, AH could promote the browning, lipolysis and thermogenesis in 3T3-L1 adipocytes and would suppress the hydrogen peroxide-induced oxidative stress and lipogenesis during differentiation. We therefore suggest that AH could be used as a potential candidate for treating obesity and related metabolic disorders.

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Choa Park, Joonwoo Park, Myeong Kuk Shim, Mee-Ra Rhyu, Byung-Koo Yoon, Kyung Sook Kim, and YoungJoo Lee

Atherosclerosis is the most common root cause of arterial disease, such as coronary artery disease and carotid artery disease. Hypoxia is associated with the formation of macrophages and increased inflammation and is known to be present in lesions of atherosclerotic. Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessels, and hypoxic conditions affect VSMC inflammation, proliferation and migration, which contribute to vascular stenosis and play a major role in the atherosclerotic process. Estrogen receptor (ER)-β is thought to play an important role in preventing the inflammatory response in VSMCs. In this report, we studied the anti-inflammatory effect of indazole (In)-Cl, an ERβ-specific agonist, under conditions of hypoxia. Expression of cyclooxygenase-2 reduced by hypoxia was inhibited by In-Cl treatment in VSMCs, and this effect was antagonized by an anti-estrogen compound. Additionally, the production of reactive oxygen species induced under conditions of hypoxia was reduced by treatment with In-Cl. Increased cell migration and invasion by hypoxia were also dramatically decreased following treatment with In-Cl. The increase in cell proliferation following treatment with platelet-derived growth factor was attenuated by In-Cl in VSMCs. RNA sequencing analysis was performed to identify changes in inflammation-related genes following In-Cl treatment in the hypoxic state. Our results suggest that ERβ is a potential therapeutic target for the suppression of hypoxia-induced inflammation in VSMCs.

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Xin-wei Chen, Ye-hong Li, Meng-jun Zhang, Zhou Chen, Dian-shan Ke, Ying Xue, and Jian-ming Hou

Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in promoting bone formation and inhibiting bone resorption; however, its effects on senile osteoporosis remain unknown. This study aimed to investigate the effects and mechanism of LF intervention using a senile osteoporosis model (SAMP6 mice) and senescent osteoblasts. Micro-CT and hematoxylin and eosin staining demonstrated that the intragastric administration (2 g/kg/day) of LF could improve the bone mass and microstructure of SAMP6 mice. Furthermore, LF treatment improved bone metabolism and increased insulin-like growth factor 1 (Igf1) mRNA expression and activated phosphorylation status of AKT. Using osteoblasts passaged for ten generations as an in vitro senescence model, various markers associated with osteoblast formation and differentiation, as well as related indices of oxidative stress were analyzed. Our results revealed that after multiple generations, osteoblasts entered senescence, in conjunction with increased oxidative stress damage, reduced bone metabolism and enhanced expression of aging-related markers. While inhibiting oxidative stress, LF improved osteoblast proliferation by promoting the expression of osteogenesis markers, including alkaline phosphatase (ALP) activity, Igf1, bone gla protein (Bglap) and osteoprotegerin/receptor activator of nuclear factor-kB ligand (Opg/Rankl) mRNA and delayed senescence by decreasing the level of p16 and p21 expression. RNAI-mediated downregulation of IGF1 attenuated the effect of LF on osteogenesis. Therefore, the findings of the present study indicate that LF may promote osteogenesis via IGF1 signaling, thereby preventing senile osteoporosis.

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Danrong Ye, Yang Jiang, Yihan Sun, Yuefeng Li, Yefeng Cai, Qingxuan Wang, Ouchen Wang, Endong Chen, and Xiaohua Zhang

Thyroid cancer is associated with one of the most malignant endocrine tumors. However, molecular mechanisms underlying thyroid tumorigenesis and progression remain unclear. In order to investigate these mechanisms, we performed whole-transcriptome sequencing, which indicated that a differentially expressed gene, METTL7B, was highly expressed in thyroid cancers. We analyzed METTL7B expression using TCGA and performed qRT-PCR on tissue samples. Moreover, an analysis of clinicopathological characteristics revealed a positive correlation between METTL7B and lymph node metastasis. A series of in vitro experiments indicated that METTL7B enhanced migration and invasion of thyroid carcinoma cells. Further studies revealed that METTL7B may enhance TGF-β1-induced epithelial-mesenchymal transition (EMT). Our results indicate that METTL7B may promote metastasis of thyroid cancer through EMT and may therefore be considered as a potential biomarker for the diagnosis and prognosis of thyroid carcinoma.

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Gabriela Silva Monteiro de Paula, Marianna Wilieman, Karina Ribeiro Silva, Leandra Santos Baptista, Sihem Boudina, Luana Lopes de Souza, Thais Bento-Bernardes, Karina Dutra Asensi, Regina Coeli dos Santos Goldenberg, and Carmen Cabanelas Pazos-Moura

Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33–39%), reduced cell proliferation at day 3 (−53%) and reduced lipid accumulation at day 21 (−86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.

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Soojin Kim, Daksh Thaper, Samir Bidnur, Paul Toren, Shusuke Akamatsu, Jennifer L Bishop, Colin Colins, Sepideh Vahid, and Amina Zoubeidi

Neuroendocrine (NE) differentiation of advanced prostate adenocarcinoma following androgen receptor (AR) axis-directed therapy is becoming increasingly recognized. Several models of this transdifferentiation provide insight into its molecular pathogenesis and have highlighted the placental gene PEG10 for further study. Using our unique model of enzalutamide resistance (ENZR) and NE differentiation, we studied PEG10/AR interplay in enzalutamide treatment-resistant cell lines 42DENZR and 42FENZR compared to LNCaP and castration-resistant 16DCRPC cells. ENZR cell lines with positive terminal NE marker status also displayed higher baseline expression of PEG10 compared to LNCaP and 16DCRPC. Antagonism of AR activity increased PEG10 expression followed by an increase in terminal NE markers. Conversely, stimulating AR activity via androgen supplementation reversed PEG10 and NE marker expression in a time and dose-dependent manner. These results were supported by human data showing that PEG10 expression is highest in NEPC and that AR-dependent gene, PSA, is negatively correlated with PEG10 in adenocarcinoma. Further, ChIP assay confirmed binding of activated AR to the PEG10 enhancer, decreasing PEG10 expression. While PEG10 did not drive NEPC, its knockdown reduced NE markers in our cell lines. Moreover, PEG10 knockdown in vitro- and in vivo-attenuated tumor growth. Overall, these observations indicate that PEG10 is an AR-repressed gene which modulates NE markers in ENZR cells and targeting PEG10 in advanced prostate cancer with NE features is a rational and viable option.

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Norman G Nicolson, Reju Korah, and Tobias Carling

Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.

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Nicola J Smith and Tim R Fenton

The interaction between human papillomaviruses (HPV) and the apolipoprotein-B mRNA-editing catalytic polypeptide-like (APOBEC)3 (A3) genes has garnered increasing attention in recent years, with considerable efforts focused on understanding their apparent roles in both viral editing and in HPV-driven carcinogenesis. Here, we review these developments and highlight several outstanding questions in the field. We consider whether editing of the virus and mutagenesis of the host are linked or whether both are essentially separate events, coincidentally mediated by a common or distinct A3 enzymes. We discuss the viral mechanisms and cellular signalling pathways implicated in A3 induction in virally infected cells and examine which of the A3 enzymes might play the major role in HPV-associated carcinogenesis and in the development of therapeutic resistance. We consider the parallels between A3 induction in HPV-infected cells and what might be causing aberrant A3 activity in HPV-independent cancers such as those arising in the bladder, lung and breast. Finally, we discuss the implications of ongoing A3 activity in tumours under treatment and the therapeutic opportunities that this may present.

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Matias Knuuttila, Esa Hämäläinen, and Matti Poutanen

Recent development of gas chromatography and liquid chromatography-tandem mass spectrometry (GC-MS/MS, LC-MS/MS) has provided novel tools to define sex steroid concentrations. These new methods overcome several of the problems associated with immunoassays for sex steroids. With the novel MS-based applications we are now able to measure small concentrations of the steroid hormones reliably and with high accuracy in both body fluids and tissue homogenates. The sensitivity of the tandem mass spectrometry assays allows us also for the first time to reliably measure picomolar or even femtomolar concentrations of estrogens and androgens. Furthermore, due to a high sensitivity and specificity of MS technology, we are also able to measure low concentrations of steroid hormones of interest in the presence of pharmacological concentration of other steroids and structurally closely related compounds. Both of these features are essential for multiple preclinical models for prostate cancer. The MS assays are also valuable for the simultaneous measurement of multiple steroids and their metabolites in small sample volumes in serum and tissue biopsies of prostate cancer patients before and after drug interventions. As a result, novel information about steroid hormone synthesis and metabolic pathways in prostate cancer has been obtained. In our recent studies, we have extensively applied a GC-MS/MS method to study androgen biosynthesis and metabolism in VCaP prostate cancer xenografts in mice. In the present review, we shortly summarize some of the benefits of the GC-MS/MS and novel LC-MS/MS assays, and provide examples of their use in defining novel mechanisms of androgen action in prostate cancer.