Cytochrome c oxidase IV isoform 1 (COX4-1) regulates the proliferation, migration and invasion of trophoblast cells via modulating mitochondrial function.

INTRODUCTION: Spontaneous miscarriage is a common complication of early pregnancy. Previous studies have shown that mitochondrial function plays an important role in establishment of a successful pregnancy. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1), a component of electron transport chain complex Ⅳ, is required for coupling the rate of ATP production to energetic requirements. However, there is very limited research on its role in trophoblast biology and how its dysfunction may contribute to spontaneous miscarriage. METHODS: Placental villi (7-10 weeks gestational age) collected from either induced termination of pregnancy or after spontaneous miscarriage were examined for expression of COX4I1. COX4I1 was knocked down by siRNA transfection of primary isolates of EVT cells. Real-time cell analysis (RTCA) and 5-Ethynyl-2'-deoxyuridine (EdU) were used to detect changes in proliferation ability after COX4I1 knockdown of EVT cells. Migration and invasion indices were determined by RTCA. Mitochondrial morphology was observed via MitoTracker staining. Oxidative phosphorylation, ATP production, and glycolysis in COX4I1-deficient cells and controls were assessed by a cellular energy metabolism analyzer (Seahorse). RESULTS: In placental villous tissue, COX4I1 expression was significantly decreased in the spontaneous miscarriage group. Knockdown of COX4I1 inhibited EVT cell proliferation, increased the migration and invasion ability and mitochondrial fusion of EVT cells. Mitochondrial respiration and glycolysis were impaired in COX4I1-deficient EVT cells. Knockdown of MMP1 could rescue the increased migration and invasion induced by COX4I1 silencing. DISCUSSION: Low expression of COX4I1 leads to mitochondrial dysfunction in EVT, resulting in altered trophoblast function, and ultimately to pregnancy loss.

Interleukin-1 beta signals through the ERK signalling pathway to modulate human placental trophoblast migration and invasion in the first trimester of pregnancy.

INTRODUCTION: Interleukin-1 beta (IL-1ß) can promote cell migration, invasion and metastasis in various cancer cells. The mechanism of its role in human trophoblast has not been fully investigated. Therefore, we aimed to investigate the expression level of IL-1ß in first trimester decidua and placenta and its potential role in regulation of extravillous trophoblast cell (EVT) invasion and migration. METHODS: First trimester placenta and decidua were collected to study the expression levels of IL-1ß and its receptors by immunohistochemical staining. Primary isolates of first trimester EVT or the HTR-8/SVneo trophoblast like cell line were used to assess migration and invasion. Matrix metalloproteinase levels were assessed by gelatin zymography and ELISA. The phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. Differentially expressed proteins in cells was detected and verified by Western Blot. RESULTS: IL-1ß, its receptors and antagonist are expressed in first trimester placenta and decidua, exogenous IL-1ß stimulates trophoblast cell outgrowth, migration and invasion through the ERK signaling pathway. IL-1ß was significantly increased in the placenta at 6-7 weeks gestation compared with 8-9 weeks gestation (P < 0.0001). Transwell and RTCA assays indicated that IL-1ß stimulates the invasion and migration of EVT. In addition, IL-1ß promoted the phosphorylation of ERK 1/2. It also promoted the expression of MMP2 and MMP9 in EVT as demonstrated by gelatin zymography assay and enzyme linked immunosorbent assay. DISCUSSION: This study demonstrated IL-1ß expression in placenta and decidua, and that it regulates EVT invasion and migration.

Extravillous trophoblast cell-derived exosomes induce vascular smooth muscle cell apoptosis via a mechanism associated with miR-143-3p.

The remodeling of uterine spiral arteries is a complex process requiring the dynamic action of various cell types. During early pregnancy, extravillous trophoblast (EVT) cells differentiate and invade the vascular wall, replacing the vascular smooth muscle cells (VSMCs). Several in vitro studies have shown that EVT cells play an important role in promoting VSMC apoptosis, however, the mechanism underlying this process is not fully understood. In this study, we demonstrated that EVT-conditioned media and EVT-derived exosomes could induce VSMC apoptosis. Through data mining and experimental verification, it was demonstrated that the EVT exosome miR-143-3p induced VSMC apoptosis in both VSMCs and a chorionic plate artery (CPA) model. Furthermore, FAS ligand was also expressed on the EVT exosomes and may play a co-ordinated role in apoptosis induction. These data clearly demonstrated that VSMC apoptosis is mediated by EVT-derived exosomes and their cargo of miR-143-3p as well as their cell surface presentation of FASL. This finding increases our understanding of the molecular mechanisms underlying the regulation of VSMC apoptosis during spiral artery remodeling.

Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

BACKGROUND: Heat shock protein 60 (HSP60) is essential for the folding and assembly of newly imported proteins to the mitochondria. HSP60 is overexpressed in most types of cancer, but its association with ovarian cancer is still in dispute. SKOV3 and OVCAR3 were used as experimental models after comparing the expression level of mitochondrial HSP60 in a normal human ovarian epithelial cell line and four ovarian cancer cell lines. RESULTS: Low HSPD1 (Heat Shock Protein Family D (HSP60) Member 1) expression was associated with unfavorable prognosis in ovarian cancer patients. Knockdown of HSPD1 significantly promoted the proliferation and migration of ovarian cancer cells. The differentially expressed proteins after HSPD1 knockdown were enriched in the lipoic acid (LA) biosynthesis and metabolism pathway, in which mitochondrial 3-oxoacyl-ACP synthase (OXSM) was the most downregulated protein and responsible for lipoic acid synthesis. HSP60 interacted with OXSM and overexpression of OXSM or LA treatment could reverse proliferation promotion mediated by HSPD1 knockdown. CONCLUSIONS: HSP60 interacted with OXSM and maintained its stability. Knockdown of HSPD1 could promote the proliferation and migration of SKOV3 and OVCAR3 via lowering the protein level of OXSM and LA synthesis.

Ezrin accelerates breast cancer liver metastasis through promoting furin-like convertase-mediated cleavage of Notch1.

BACKGROUND: Ezrin, known as a crosslinker between the plasma membrane and actin cytoskeleton, is closely associated with breast cancer (BC) progression. Here, we explored a novel role of ezrin in breast cancer liver metastasis (BCLM). METHODS: The clinical relevance of ezrin was evaluated using in silico tools and confirmed in BC specimens. The effect of ezrin on proliferation, migration and invasion was examined in vitro and in vivo using murine primary liver-metastatic breast cancer cells (mLM). The molecular mechanism involved in ezrin-mediated activation of the Notch1 signaling pathway was elucidated using in vitro models. RESULTS: Data-mining demonstrated that ezrin mRNA and protein expression is up-regulated in breast cancer cohorts and has prognostic significance. Ezrin overexpression promotes cell proliferation, migration and invasion in vitro and in vivo. Hairy and enhancer of split-1 (Hes1) is one of the most significantly enriched candidates of differentially expressed genes in ezrin overexpression and control mLM cells. Ezrin can positively regulate Hes1 mRNA and protein expression, and their coexpression was associated with poor prognosis in BC patients. Ezrin promoted BC cell proliferation in a Hes1-dependent manner without directly interacting with Hes1. The functional link between ezrin and Hes1 is dependent on Notch1 activation through promotion of furin-like convertase cleavage. CONCLUSION: Our results demonstrated that ezrin drives BCLM through activation of the Notch signaling pathway via furin-like convertase. These findings provide a better understanding of the mechanism of ezrin in breast cancer progression, with the goal of discovering a novel target for the treatment of BCLM in the future.

Contribution of vascular smooth muscle cell apoptosis to spiral artery remodeling in early human pregnancy.

Uterine spiral artery remodeling is one of the key maternal adaptations of pregnancy, allowing delivery of the large volumes of maternal blood required for both placental and fetal growth. Failure of this process is associated with obstetric complications including preeclampsia, fetal growth restriction and miscarriage. Spiral artery remodeling is characterized by loss of the musculoelastic wall which is replaced by fibrinoid and intramural extravillous trophoblast cells (EVT). In recent years attention has focused on the initial stages of spiral artery remodeling which include separation of the vascular smooth muscle cells (VSMCs) and their phenotypic switch to a more synthetic phenotype, facilitating their migration away from the vessel wall. However, less is known about the final fate of the VSMCs. In vitro studies suggested that EVT could induce VSMC apoptosis, though VSMC apoptosis is not seen within the wall of the spiral arteries undergoing remodeling. However, apoptotic VSMCs have been observed amongst those cells which had migrated away from the vessel wall. This review article gives a brief overview of the current state of knowledge of the processes involved in spiral artery remodeling, and then concentrates on the mechanisms involved in VSMC apoptosis, drawing on knowledge from other vascular beds.

Decidual macrophage derived MMP3 contributes to extracellular matrix breakdown in spiral artery remodeling in early human pregnancy.

Remodeling of the uterine spiral arteries is required for a successful pregnancy. This process requires the co-ordinated activity of a number of different cell types including uterine natural killer cells, decidual macrophages, extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells. We have previously demonstrated that decidual macrophages facilitate breakdown of fibronectin and laminin in a model of spiral artery remodeling. The aim of the current study was to determine which matrix metalloproteinases (MMPs) decidual macrophages express and play roles in extracellular matrix (ECM) breakdown in vascular remodeling. Decidual macrophages were isolated from first trimester decidua and cultured for 24 h to obtain conditioned medium. MMP secretion was assessed by a membrane based array and immunohistochemistry of decidual sections. In addition, the chorionic plate artery (CPA) model was used with decidual macrophage conditioned medium, with and without a MMP3 inhibitor and ECM protein expression assessed using quickscore. The decidual macrophages secreted a wide range of MMPs, with MMP3 being the most predominant. Co-localization of MMP3 to decidual macrophages was confirmed by immunohistochemistry. Decidual macrophage conditioned medium facilitated breakdown of laminin and fibronectin in the CPA model, an effect that was abrogated by the MMP3 inhibitor. These data further support the role of decidual macrophages in tissue remodeling in the first trimester of pregnancy. An alteration in their numbers or phenotype would impact spiral artery remodeling and contribute to the etiology of a number of complications of pregnancy.

The EGFR-P38 MAPK axis up-regulates PD-L1 through miR-675-5p and down-regulates HLA-ABC via hexokinase-2 in hepatocellular carcinoma cells.

BACKGROUND: Immunotherapy has been shown to be a promising strategy against human cancers. A better understanding of the immune regulation in hepatocellular carcinoma (HCC) could help the development of immunotherapy against HCC. The epidermal growth factor receptor (EGFR) signaling is frequently activated in HCC and plays important roles in tumorigenesis. However, its role in HCC immunity is still largely unknown. This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1 (PD-L1) and human leukocyte antigen class-I (HLA-I) expression in HCC cells and its underlying mechanisms. METHODS: The expression of phosphorylated EGFR (p-EGFR), PD-L1, and HLA-I (HLA-ABC) in HCC specimens was detected by immunohistochemistry, and their correlations were analyzed. PD-L1 and HLA-ABC expression in EGFR-activated HCC cells were detected by quantitative real-time PCR, Western blotting, and flow cytometry, and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells. Furthermore, the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments, luciferase reporter assay, and gene gain- and loss-of-function studies. RESULTS: p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs. EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells, which was functionally important and could be abolished by the EGFR inhibitor, gefitinib, both in vitro and in vivo. Mechanistically, enhanced P38 mitogen-activated protein kinase (MAPK) activation down-regulated microRNA-675-5p (miR-675-5p) and up-regulated glycolysis-related enzyme hexokinase 2 (HK2); miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3'-untranslated region (3'-UTR) of PD-L1 and thereby caused PD-L1 accumulation, and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC. CONCLUSIONS: The EGFR-P38 MAPK axis could up-regulate PD-L1 through miR-675-5p and down-regulate HLA-ABC via HK2 in HCC cells. Our study reveals a novel signaling network that may cause immune suppression in HCC and suggests that EGFR signaling can be targeted for HCC immunotherapy.

Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling.

Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.

Garcinol inhibits the proliferation of endometrial cancer cells by inducing cell cycle arrest.

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer­related deaths in women worldwide. The long­term survival rate is lower in advanced­stage and recurrent EC, therefore it is important to identify new anticancer drugs. Garcinol, a polyisoprenylated benzophenone, is a promising anticancer drug for various cancer types but its effects on EC remain unclear. To investigate the anticancer effects of garcinol on EC, cell proliferation and cell cycle were assessed by real­time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5­ethynyl­2'­deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC­1B cell lines. Western blotting was used to evaluate the expression of cell cycle­related protein cyclins, cyclin­dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC­1B cell proliferation in a dose­dependent manner, and induced ISH and HEC­1B cell cycle arrest at the G1 phase and G2/M phase, respectively, and decreased the S phase and DNA synthesis in these two cell lines. Following garcinol treatment the expression levels of p53 and p21 were increased, while the expression levels of CDK2, CDK4, cyclin D1 and cyclin B1 were gradually decreased in a dose­dependent manner in both ISH and HEC­1B cells. In addition, the expression levels of phosphorylated c­JUN N­terminal kinase (JNK) and p­c­JUN were significantly increased in both types of cells. Collectively, garcinol can induce EC cell cycle arrest and may be a promising candidate for EC chemotherapy.

Blockade of OX40/OX40L pathway combined with ethylene-carbodiimide-fixed donor splenocytes induces donor-specific allograft tolerance in presensitized recipients.

BACKGROUND: Memory T cells (Tms) are the major barrier preventing long-term allograft survival in presensitized transplant recipients. The OX40/OX40L pathway is important in the induction and maintenance of Tms. METHODS: In this study, we added anti-OX40L mAb to ethylene-carbodiimide-fixed donor splenocytes (ECDI-SPs)-a method which is effective in inducing allograft tolerance in non-presensitized mouse heart transplant model. Recipient mice received heart transplantation after 6 weeks of donor skin presensitization and were treated with anti-OX40L mAb, ECDI-SPs or anti-OX40L mAb + ECDI-SPs, respectively. RESULTS: Our data showed that the combination of ECDI-SPs and anti-OX40L mAb induced donor-specific tolerance in skin-presensitized heart transplant recipients, with the mechanism for this being associated with suppression of Tms and upregulation of CD4+CD25+Foxp3+ T regulatory cells (Tregs). Importantly, CD25+ T-cell depletion in the combined therapy-treated recipients broke the establishment of allograft tolerance, whereas adoptive transfer of presensitization-derived T cells into tolerant recipients suppressed Tregs expansion and abolished established tolerance. CONCLUSIONS: Blockade of OX40/OX40L pathway in combination with ECDI-SPs appears to modulate the Tms/Tregs imbalance so as to create a protective milieu and induce graft tolerance in presensitized recipients.

Vitamin D Promotes Trophoblast Cell Induced Separation of Vascular Smooth Muscle Cells in Vascular Remodeling via Induction of G-CSF.

Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA) ex vivo model of vascular remodeling to determine the effects of 1,25(OH)2D treated uNK cell, placental explant (PEx), and uNK/PEx conditioned medium (CM) on vascular smooth muscle cell (VSMC) disorganization and phenotypic switching. Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.

T-cadherin inhibits invasion and migration of endometrial stromal cells in endometriosis.

STUDY QUESTION: What is the expression level of T-cadherin in endometriosis, and does T-cadherin play a role in regulating invasion and migration of endometrial stromal cells? SUMMARY ANSWER: T-cadherin expression was reduced in ectopic endometriotic lesions compared to eutopic endometrium, and T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. WHAT IS KNOWN ALREADY: Endometriosis is a disease that involves active cell invasion and migration. T-cadherin can inhibit cell invasion, migration and proliferation in various cancer cells, but its role in endometriosis has not been investigated. STUDY DESIGN, SIZE, DURATION: We explored the expression status of T-cadherin in 40 patients with and 24 without endometriosis. We also isolated endometrial stromal cells to study the invasion, migration and signaling pathway regulation of T-cadherin overexpression. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were recruited at the Guangzhou Women and Children's Medical Center to study the expression levels of T-cadherin. The expression of T-cadherin was detected by immunohistochemistry staining and western blot. H-score was used to evaluate the staining intensity of T-cadherin. The correlation between T-cadherin expression levels (H-score) and endometriosis patients' age, stage, lesion size and adhesion was analyzed. Endometrial stromal cells from patients with and without endometriosis were isolated, and cell invasion and migration were detected by transwell assays after T-cadherin overexpression. The expression of vimentin in T-cadherin-overexpressed cells was detected by western blot. After T-cadherin overexpression, the phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference in the expression of T-cadherin in the normal endometrium of control patients and the eutopic endometrium of endometriotic patients, but it was significantly decreased in the ectopic endometrium of endometriotic patients, compared with control endometrium and eutopic endometrium of endometriosis patients (P < 0.0001, for both). Western blot analysis also showed that the expression of T-cadherin was decreased in ectopic endometriotic lesions, but not the normal control endometrium or the endometriotic eutopic endometrium. The results of transwell assays indicated that T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. In addition, T-cadherin overexpression promoted the phosphorylation of HSP27 (S78/S82) and JNK 1/2/3 (T183/Y185, T221/Y223) and decreased the expression of vimentin, MMP2 and MMP9 in eutopic endometriosis stromal cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The control group were patients with benign gynecological conditions (e.g. uterus myoma, endometrial or cervical polyp), which may have genetic or epigenetic variations associated with T-cadherin expression and signaling pathways. The case numbers of involved endometriosis and control patients were limited. This study only used endometrial stromal cells from patients with or without endometriosis. Ideally, ectopic endometrial stromal cells of the ovarian endometriotic lesions should also be utilized to explore the function of T-cadherin. WIDER IMPLICATIONS OF THE FINDINGS: Further investigation of the role of T-cadherin in endometriosis may generate new potential therapeutic targets for this complex disorder. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by the Natural Science Foundation of Guangdong Province (2016A030313495), National Natural Science Foundation of China (81702567, 81671406, 31871412), the Science and Technology Programs of Guangdong (2017A050501021), Medical Science Technology Research Fund of Guangdong Province (A2018075), the Science and Technology Programs of Guangzhou City (201704030103), Internal Project of Family Planning Research Institute of Guangdong Province (S2018004), Post-doc initiation fund of Guangzhou (3302) and Post-doc science research initiation fund of Guangzhou Women and Children's Medical Center (20160322). There are no conflicts of interest.

Understanding and management of gestational trophoblastic disease.

Gestational trophoblastic disease or neoplasia covers a spectrum of benign and malignant conditions arising from pregnancies with highly abnormal development of trophoblastic tissue. In this brief review, we discuss the different features of these different conditions and their origins and risk factors and introduce some of the more novel and controversial treatment options currently being explored.

EGF is highly expressed in hepatocellular carcinoma (HCC) and promotes motility of HCC cells via fibronectin.

Better understanding of metastasis process would allow for the development of effective approaches to treat hepatocellular carcinoma (HCC). Recent literature has highlighted the fundamental role of interaction between tumor cells and their microenvironment components in tumor metastasis. Aberrant expression of epidermal growth factor (EGF) induces highly malignant HCC, and activated EGF/EGFR signaling is correlated with an aggressive phenotype and intrahepatic metastasis. Thus, EGF in the tumor microenvironment may influence the behavior of HCC cells. In this study, for the first time, we studied the expression of EGF in HCCs, and the potential role of EGF in the motility of HCC cells and the underlying mechanisms. It was demonstrated that EGF was highly expressed in HCCs and positively associated with higher tumor grade. In addition, EGF promoted the migration and invasion of HCC cells mainly via induction of fibronectin (FN) in vitro. Mechanistically, EGF simultaneously increased the nuclear translocation and PKC mediated phosphorylation of p65 which could bind to the -356 bp to -259 bp fragment of FN promoter, leading to a markedly increased activity of FN promoter in HCC cells. These results highlight the potential role of EGF in promoting HCC metastasis, demonstrate a novel pathway for regulation of FN expression and provide potential targets for HCC prevention and treatment.

Participation of CCL1 in Snail-Positive Fibroblasts in Colorectal Cancer Contribute to 5-Fluorouracil/Paclitaxel Chemoresistance.

PURPOSE: Cancer-associated fibroblasts (CAFs) activated by cancer cells has a central role in development and malignant biological behavior in colorectal cancer (CRC). Adult fibroblasts do not express Snail, but Snail-positive fibroblasts are discovered in the stroma of malignant CRC and reported to be the key role to chemoresistance. However, the reciprocal effect of CAFs expressed Snail to chemoresistance on CRC cells and the underlying molecular mechanisms are not fully characterized. MATERIALS AND METHODS: Snail-overexpressed 3T3 stable cell lines were generated by lipidosome and CT26 mixed with 3T3-Snail subcutaneous transplanted CRC models were established by subcutaneous injection. Cell Counting Kit-8, flow cytometry and western blotting assays were performed, and immunohistochemistry staining was studied. The cytokines participated in chemoresistance was validated with reverse transcriptase-polymerase chain reaction and heatmap. RESULTS: Snail-expression fibroblasts are discovered in human and mouse spontaneous CRCs. Overexpression of Snail induces 3T3 fibroblasts transdifferentiation to CAFs. CT26 co-cultured with 3T3-Snail resisted the impairment from 5-fluorouracil and paclitaxel in vitro. The subcutaneous transplanted tumor models included 3T3-Snail cells develop without restrictions even after treating with 5-fluorouracil or paclitaxel. Moreover, these chemoresistant processes may be mediated by CCL1 secreted by Snail-expression fibroblasts via transforming growth factor ß/nuclear factor-κB signaling pathways. CONCLUSION: Taken together, Snail-expressing 3T3 fibroblasts display CAFs properties that support 5-fluorouracil and paclitaxel chemoresistance in CRC via participation of CCL1 and suggest that inhibition of the Snail-expression fibroblasts in tumor may be a useful strategy to limit chemoresistance.

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis.

Maize is one of the most widely cultivated crops. It accumulates a large quantity of seed storage proteins, which are important for seed development and germination, and contribute to the nutritional quality of seeds. Based on solubility, the storage proteins are divided into albumins (water-soluble), globulins (salt-soluble), prolamins (alcohol-soluble), and glutelins (acid- or alkali-soluble). Maize hybrids are cultivated due to the superior performance of F1 hybrids than that of their parents, a phenomenon known as heterosis. However, the accumulation patterns of seed storage proteins in maize embryos between the hybrids and their parental inbred lines have not been compared. In the present study, two elite inbred lines of China, Zheng 58 and Chang 7-2, and their reciprocal hybrids (Zheng 58 × Chang 7-2 and Chang 7-2 × Zheng 58) were used to explore parental influences on the accumulation patterns of seed storage proteins in maize embryos. For this purpose, we focused on seed salt-soluble proteins (SSPs) in our experiments. The SSPs were selectively extracted from maize mature embryos after extensive removal of water-soluble albumin and separated using two-dimensional gel electrophoresis (2-DE), followed by mass spectrometry analysis. Our results indicated that the 2-DE SSP profiles of hybrids closely resembled those of their maternal parent rather than the paternal parent. In other words, 2-DE SSP profiles of Zheng 58 × Chang 7-2 were more similar those of Zheng 58 whereas such profiles of Chang 7-2 × Zheng 58 were more similar to those of Chang 7-2 although the 2-DE profiles of all four maize types were quite similar. In total, 12 relatively abundant SSPs spots representing five kinds of proteins were identified, of which nine protein spots displayed non-additive accumulation in at least one hybrid. This study provided additional data on dominance and partial dominance effects on maize hybrids embryos. Besides, earlier studies on accumulation profiles of globulin-1 (also known as vicilin), which is one of the most abundant globulins in maize embryos, also support the above results. This study would be helpful in revealing the mechanisms underlying SSPs accumulation patterns in the hybrids.

Epidermal growth factor and tumor necrosis factor α cooperatively promote the motility of hepatocellular carcinoma cell lines via synergistic induction of fibronectin by NF-κB/p65.

BACKGROUND: The interaction between hepatocellular carcinoma (HCC) cells and their microenvironment plays a fundamental role in tumor metastasis. The HCC microenvironment is rich in epidermal growth factor (EGF) and tumor necrosis factor α (TNFα), which may cooperatively, rather than individually, interact with tumor cells to influence their biological behavior. METHODS: Immunohistochemistry was performed to study the expression of EGF and TNFα in HCCs. Western blotting, immunofluorescence, qRT-PCR, wound healing scratch and invasion assay, and chromatin immunoprecipitation assays were used to study the combined roles of EGF and TNFα in the motility of HCC cells in vitro. RESULTS: We demonstrated that both EGF and TNFα were highly expressed in HCCs, and HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. In vitro, EGF and TNFα cooperatively promoted the motility of HCC cells mainly via synergistic induction of an extracellular matrix glycoprotein fibronectin (FN). Mechanistically, EGF and TNFα jointly increased the nuclear translocation and PKC mediated phosphorylation of NF-κB/p65 which could bind to the -356bp to -259bp fragment of the FN promoter, leading to a markedly increased activity of the FN promoter in HCC cells. CONCLUSIONS: HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. EGF and TNFα cooperatively promoted the motility of HCC cells mainly through NF-κB/p65 mediated synergistic induction of FN in vitro. GENERAL SIGNIFICANCE: These findings highlight the crosstalk between EGF and TNFα in promoting HCC, and provide potential targets for HCC prevention and treatment.