Screening for metastasis-related genes in mouse melanoma cells through sequential tail vein injection.

Tumor metastasis, responsible for approximately 90% of cancer-associated mortality, remains poorly understood. Here in this study, we employed a melanoma lung metastasis model to screen for metastasis-related genes. By sequential tail vein injection of mouse melanoma B16F10 cells and the subsequently derived cells from lung metastasis into BALB/c mice, we successfully obtained highly metastatic B16F15 cells after five rounds of in vivo screening. RNA-sequencing analysis of B16F15 and B16F10 cells revealed a number of differentially expressed genes, some of these genes have previously been associated with tumor metastasis while others are novel discoveries. The identification of these metastasis-related genes not only improves our understanding of the metastasis mechanisms, but also provides potential diagnostic biomarkers and therapeutic targets for metastatic melanoma.

The roles and mechanism of STIM1 in tumorigenesis and metastasis.

STIM1 (stromal interaction molecule 1) is one of the key components of the store operated Ca2+ entry channel (SOCE), which is located on the endoplasmic reticulum membrane and highly expressed in most kinds of tumors. STIM1 promotes tumorigenesis and metastasis by modulating the formation of invadopodia, promoting angiogenesis, mediating inflammatory response, altering the cytoskeleton and cell dynamics. However, the roles and mechanism of STIM1 in different tumors have not been fully elucidated. In this review, we summarize the latest progress and mechanisms of STIM1 in tumorigenesis and metastasis, thereby providing insights and references for the study on STIM1 in the field of cancer biology in the future.

The role of hepatocyte nuclear factor 4α (HNF4α) in tumorigenesis.

Hepatocyte Nuclear Factor 4 Alpha (HNF4α) is a master transcription factor mainly expressed in the liver, kidney, intestine and endocrine pancreas. It regulates multiple target genes involved in embryonic development and metabolism. HNF4α-related diseases include non-alcoholic fatty liver disease (NAFLD), obesity, hypertension, hyperlipidemia, metabolic syndrome and diabetes mellitus. Recently, HNF4α has been emerging as a key player in a variety of cancers. In this review, we summarized the role and mechanism of HNF4α in different types of cancers, especially in liver and colorectal cancer, aiming to provide additional guidance for intervention of these diseases.

PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas.

OBJECTIVE: The BRAF inhibitor, vemurafenib, has been widely used in the treatment of patients with melanoma-bearing BRAFV600E mutations. While the initial response to vemurafenib is usually excellent, the majority of patients eventually develop resistance and metastatic disease. However, the underlying molecular mechanism remains elusive. The objective of this study was therefore to identify additional molecular targets responsible for vemurafenib resistance. METHODS: Western blots and immunohistochemistry analyses were used to evaluate expressions of PYK2 and p-PYK2 in cultured cells and melanoma tissue microarrays. The relationships of p-PYK2 with clinicopathological parameters were statistically analyzed. Invadopodia cell invasion, and a Ca2+ assay were used to determine the effect of vemurafenib resistance-induced p-PYK2 on melanoma progression. A mouse model was used to assess the effects of PYK2 on melanoma metastasis. RESULTS: Elevated p-PYK2 levels were detected in vemurafenib-resistant melanoma cells, and PYK2 was shown to regulate invadopodia formation in melanoma cells. Vemurafenib triggered invadopodia formation by activation of PYK2. Inhibition of PYK2 with either shRNA or the small molecule inhibitor, PF562711, dramatically reduced vemurafenib-induced invadopodia formation. Furthermore, knockdown of PYK2 significantly reduced melanoma lung metastasis in vivo. Increased expressions of p-PYK2 in melanoma patients were positively correlated with advanced stage (P = 0.002), metastasis (P < 0.001), and Clark grade (P < 0.001), and were also associated with short overall survival [hazard ratio (HR) = 3.304, P = 0.007] and progression-free survival (HR = 2.930, P = 0.001). CONCLUSIONS: PYK2 mediated vemurafenib-induced melanoma cell migration and invasion. Inhibition of PYK2 resensitized melanoma cells to vemurafenib. Phospho-PYK2 was a prognostic biomarker in melanoma patients.

Anthocyanin Biosynthesis and Methylation of the MdMYB10 Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple.

The color of apple skin, particularly anthocyanin-based coloration, is a key factor determining market acceptance. The mechanisms of anthocyanin accumulation in apples with different skin color patterns (i.e., striped and blushed) were analyzed. In total, 14 anthocyanins and 5 procyanidins were simultaneously assayed in red blushed-skin mutants (CF-B1 and CF-B2) and red striped-skin parents (CF-S1 and CF-S2), and 13 significant differences were revealed. Anthocyanin accumulation was significantly higher in the red blushed-skin apples than it was in the parents. The transcript levels of anthocyanin biosynthesis genes and regulatory factors (MdMYB10, MdbHLH3, and MdWD40) were associated with different skin color patterns during the coloring period at 4, 6, and 8 days after the fruits were debagged. The methylation levels of the MdMYB10 promoter regions -1203 to -779 bp, -1667 to -1180 bp, and -2295 to -1929 bp were associated with different skin color patterns, and there was more methylation in red striped-skin apples. These results improve our understanding of anthocyanin accumulation and its underlying molecular mechanism in apples with different skin color patterns, thereby providing valuable information for apple breeding.

Therapeutic sildenafil inhibits pulmonary damage induced by cigarette smoke exposure and bacterial inhalation in rats.

Context: Clinical reports showed sildenafil beneficial therapy on severe chronic obstructive pulmonary disease (COPD) with pulmonary hypertension (PH) patients.Objective: The study investigated therapeutic effects of silenafil on pulmonary damage induced by cigarette smoke exposure and bacterial inhalation in rats.Materials and methods: Female Sprague-Dawley rats (200-250 g) were divided into control group (no exposure, n = 10) and exposure group (n = 50) suffered from cigarette smoke exposure and Klebsiella pneumonia inhalation for 8 weeks. Then rats were orally given normal saline (control group or model group), 2.0, 3.0, or 4.5 mg/kg sildenafil for 4 weeks, respectively. Pulmonary pressure, RVHI and morphological analysis of pulmonary vascular remodeling, respiratory functions assay, morphological analysis of pulmonary alveoli, and expression of PCNA and caspase-3 of epithelial cells in bronchioles wall were examined.Results: Compared to model rats, 2.0, 3.0, and 4.5 mg/kg sildenafil increased VT by -0.6 to 9.58%, PEF by 3.12 to 6.49%, EF50 by 0.81 to 6.50%, decreased mPAP by 4.43 to 25.58%, RVHI by 6.54 to 26.41%, showing a dose-dependent improvement. Furthermore, 4.5 mg/kg sildenafil significantly increased MAN by 39.70%, LA/CSA by 37.07%, decreased muscular pulmonary arteries by 48.00%, WT by 12.83%, MT by 22.89%, caspase-3 expression by 17.71%, and showed improvement on abnormality in lung interstitial and bronchioles by microscopy.Discussion and conclusion: Our results demonstrated that sildenafil decreased pathological changes in alveoli, bronchioles, interstitial tissue, and arterioles of rats with COPD and PH.

One-step construction of a transition-metal surface decorated with metal sulfide nanoparticles: A high-efficiency electrocatalyst for hydrogen generation.

Sluggish water dissociation kinetics in the Volmer step is still the huge challenge in designing and synthesizing transition-metal-based electrocatalysts for hydrogen evolution reaction (HER) in alkaline media. Herein, we report a general one-step electrodeposition strategy to construct a high-efficiency electrocatalytic system based on transition metals (Ni, Co and Fe) surface decorated with metal sulfide (Ni3S2, CoxSy and FexSy) nanoparticles on Ni foam with thiourea (MxSy/M/NF). Evaluated in 1.0 M KOH, the obtained Ni3S2/Ni/NF, CoxSy/Co/NF and FexSy/Fe/NF exhibit exceptionally enhanced HER activity, whose overpotentials at 10 mA cm-2 decrease from 196 to 45 mV, 217 to 89 mV and 288 to 128 mV, respectively, when metal sulfides are introduced to Ni/NF, Co/NF and Fe/NF electrodes. Compared with most of state-of-the-art transition-metal-based catalysts, the Ni3S2/Ni/NF electrode displays extremely low overpotential and small Tafel slope (54 mV dec-1) as well as excellent stability. Further experimental characterizations and density functional theory calculations reveal that such excellent HER performance can be ascribed to the synergetic effect in the combination of metals and metal sulfides, the former of which enhances water dissociation and the latter accelerates hydrogen generation. This intriguing work opens a new avenue towards designing highly active low-cost electrocatalysts for commercial application.

Construction of CoO/Co-Cu-S Hierarchical Tubular Heterostructures for Hybrid Supercapacitors.

Hierarchical hollow structures for electrode materials of supercapacitors could enlarge the surface area, accelerate the transport of ions and electrons, and accommodate volume expansion during cycling. Besides, construction of heterostructures would enhance the internal electric fields to regulate the electronic structures. All these features of hierarchical hollow heterostructures are beneficial for promoting the electrochemical properties and stability of electrode materials for high-performance supercapacitors. Herein, CoO/Co-Cu-S hierarchical tubular heterostructures (HTHSs) composed of nanoneedles are prepared by an efficient multi-step approach. The optimized sample exhibits a high specific capacity of 320 mAh g-1 (2300 F g-1 ) at 2.0 A g-1 and outstanding cycling stability with 96.5 % of the initial capacity retained after 5000 cycles at 10 A g-1 . Moreover, an all-solid-state hybrid supercapacitor (HSC) constructed with the CoO/Co-Cu-S and actived carbon shows a stable and high energy density of 90.7 Wh kg-1 at a power density of 800 W kg-1 .

STIM1 overexpression in hypoxia microenvironment contributes to pancreatic carcinoma progression.

OBJECTIVE: Stromal interaction molecule 1 (STIM1) overexpression has been reported to play an important role in progression of several cancers. However, the mechanism of STIM1 overexpression and its relationship with hypoxia in pancreatic ductal adenocarcinoma (PDAC) remains unclear. METHODS: STIM1 and HIF-1α expression was tested using immunohistochemistry in tissue microarray (TMA) including pancreatic cancer and matched normal pancreatic tissues, and their relationships with clinicopathological parameters were statistically analyzed. q-PCR, Western blot, ChIP, and luciferase assay were employed to 030 analyze transcriptional regulation between HIF-1α and STIM1 in pancreatic cancer PANC-1 cells. RESULTS: Both STIM1 and HIF-1α showed higher positive rates and up-regulated expression in cancer tissues compared to that of normal tissues (P < 0.05). The Kaplan-Meier method revealed that higher HIF-1α and STIM1 expression levels were significantly correlated with decreased disease-free survival ( P = 0.025 and P = 0.029, respectively). The expression of HIF-1α showed a significant positive correlation with that of STIM1 in cancer tissues (rs = 0.3343, P = 0.0011) and pancreatic cancer cell lines. Furthermore, ChIP and luciferase assays confirmed that HIF-1α bound to the STIM1 promoter and regulated its expression in PANC-1 cells. CONCLUSIONS: In hypoxia microenvironment, up-regulated expression of STIM1 mediated by HIF-1α promotes PDAC progression. HIF-1α and STIM1 are potential prognostic markers and/or therapeutic targets for PDAC treatment.

A facile sequential ion exchange strategy to synthesize CoSe2/FeSe2 double-shelled hollow nanocuboids for the highly active and stable oxygen evolution reaction.

Transition metal-based nanostructures have been considered as promising substitutes for rare-earth metal oxide electrocatalysts toward the oxygen evolution reaction (OER). Herein, we report for the first time on a novel multicomponent metal selenide electrocatalyst based on CoSe2/FeSe2 double-shelled hollow nanocuboids (CoSe2/FeSe2 DS-HNCs) with the highly oxidative Co3+ species, which is synthesized via a facile sequential ion exchange strategy. The solid Co-precursor nanocuboids are first converted into the intermediate Co2[Fe(CN)6] with a mesoporous and double-shelled hollow structure produced through a facile ligand exchange at room temperature, and then the final CoSe2/FeSe2 DS-HNCs are obtained by a subsequent Se ion exchange reaction. The intermediate product of Co2[Fe(CN)6] plays an important role not only in constructing a double-shelled hollow structure but also in providing the Fe source for the growth of the final multicomponent metal selenides. Benefiting from the nanosized double-shelled hollow structure and mesoporous double-metal selenide shells with the highly oxidative Co3+ species, the as-prepared CoSe2/FeSe2 DS-HNCs exhibit superior OER performance to state-of-the-art metal selenides, including a small overpotential of 240 mV at a current density of 10 mA cm-2 and the excellent electrochemical durability over 50 h. This work opens up a new avenue towards developing highly active multicomponent noble-metal-free electrocatalysts.

Hesperidin inhibits the epithelial to mesenchymal transition induced by transforming growth factor-ß1 in A549 cells through Smad signaling in the cytoplasm

Hesperidin, a natural compound, suppresses the epithelial-to-mesenchymal transition through the TGF-ß1/Smad signaling pathway. However, studies on the detailed effects and mechanisms of hesperidin are rare. The present study showed that, for A549 alveolar epithelial cells, the anti-proliferative effects of hesperidin occurred in a dose-dependent manner, with an IC50= 216.8 µM at 48 h. TGF-ß1 was used to activate the Smad signaling pathway and induce the epithelial to mesenchymal transition in cells. Treatment with hesperidin or SB431542 was used for antagonism of Smad pathway activation. Hesperidin inhibited the increase in ɑ-SMA and Col1ɑ-1 and the decrease in E-cadherin in a dose-dependent manner from concentration of 20 µM to 60 µM, as assessed by both ELISA and Western blotting assays; however, there was no significant effect on cellular morphological alterations. Moreover, the Western blotting assay showed that, in the cytoplasm, hesperidin and SB431542 had no significant effect on the protein expression of Smad 2, 3, 4, or 7 as well as 2/3. However, 60 µM hesperidin and SB431542 significantly decreased p-Smad2/3 protein expression. From the above results, it is concluded that hesperidin can partly inhibit the epithelial to mesenchymal transition in human alveolar epithelial cells; the effect accounts for the blockage of the phosphorylation of Smad2/3 in the cytoplasm rather than a change in Smad protein production in the cytoplasm

Preprocessing with Photoshop Software on Microscopic Images of A549 Cells in Epithelial-Mesenchymal Transition.

OBJECTIVE: To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). STUDY DESIGN: Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. RESULTS: As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p < 0.01 or 0.001). The values of intraclass correlation coefficient, both in Single Type or Average, were higher in IPPs than in Controls both for 3 observers and 1 observer. CONCLUSION: Processed with Adobe Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

Suitable parameter choice on quantitative morphology of A549 cell in epithelial-mesenchymal transition.

Evaluation of morphological changes in cells is an integral part of study on epithelial to mesenchymal transition (EMT), however, only a few papers reported the changes in quantitative parameters and no article compared different parameters for demanding better parameters. In the study, the purpose was to investigate suitable parameters for quantitative evaluation of EMT morphological changes. A549 human lung adenocarcinoma cell line was selected for the study. Some cells were stimulated by transforming growth factor-ß1 (TGF-ß1) for EMT, and other cells were as control without TGF-ß1 stimulation. Subsequently, cells were placed in phase contrast microscope and three arbitrary fields were captured and saved with a personal computer. Using the tools of Photoshop software, some cells in an image were selected, segmented out and exchanged into unique hue, and other part in the image was shifted into another unique hue. The cells were calculated with 29 morphological parameters by Image Pro Plus software. A parameter between cells with or without TGF-ß1 stimulation was compared statistically and nine parameters were significantly different between them. Receiver operating characteristic curve (ROC curve) of a parameter was described with SPSS software and F-test was used to compare two areas under the curves (AUCs) in Excel. Among them, roundness and radius ratio were the most AUCs and were significant higher than the other parameters. The results provided a new method with quantitative assessment of cell morphology during EMT, and found out two parameters, roundness and radius ratio, as suitable for quantification.

[Inhibitory effect of Panax notoginseng saponins on alveolar epithelial to mesenchymal transition].

In the study, the effects of Panax notoginseng saponins (PNS) on alveolar epithelial to mesenchymal transition (EMT) and extracellular matrix degradation were observed in a type of human alveolar epithelial cell, A549 cells, stimulated by TGF-beta1. Firstly, MTT method was applied to evaluation of cellular proliferation and found that PNS from 12.5 mg x L(-1) to 200 mg x L(-1) dosage could not inhibit significantly cellular proliferation. Then, cells were divided into five groups, normal group, TGF-beta1 group, TGF-beta1 + 50 mg x L(-1) PNS group, TGF-beta1 + 100 mg x L(-1) PNS group and TGF-beta1 + 200 mg x L(-1) PNS group. Normal cells were not stimulatec by TGF-beta1; TGF-beta1 cells were only stimulated by TGF-beta1 and the other cells were stimulated by TGF-beta1 with different doses of PNS, respectively. After stimulation, cells and supernatants were collected for assays. Cellular roundness was applied to quantitative evaluation of morphological change. Immunocytochemistry was applied to examine E-cadherion, a-SMA and FN proteins expression in the cells. Enzyme linked-immunosorbent assay was applied to MMP-9 and TIMP-1 levels. The results showed that EMT of A549 cells was induced by TGF-beta1, showing significant change of roundness, E-cadherion, alpha-SMA and FN (P < 0.05, P < 0.01). Compared to TGF-beta1, PNS significantly inhibited the changes of roundness (P < 0.05), FN and alpha-SMA (P < 0.05, P < 0.01) and not significantly inhibited the change of E-cadherion. Furthermore, MMP-9 levels were significantly increased by TGFbeta1 stimulation (P < 0.05), without significant change of TIMP-1. Compared with TGF-beta1, PNS could significantly increase MMP-9 level (P < 0.05) and decrease TIMP-1 levels (P < 0.05, P < 0.01). In conclusion, PNS could inhibit alveolar epithelial cell EMT induced by TGF-beta1, with increase of extracellular matrix degradation ability, which showed anti-fibrosis of lung ability.

STIM1- and Orai1-mediated Ca(2+) oscillation orchestrates invadopodium formation and melanoma invasion.

Ca(2+) signaling has been increasingly implicated in cancer invasion and metastasis, and yet, the underlying mechanisms remained largely unknown. In this paper, we report that STIM1- and Orai1-mediated Ca(2+) oscillations promote melanoma invasion by orchestrating invadopodium assembly and extracellular matrix (ECM) degradation. Ca(2+) oscillation signals facilitate invadopodial precursor assembly by activating Src. Disruption of Ca(2+) oscillations inhibited invadopodium assembly. Furthermore, STIM1 and Orai1 regulate the proteolysis activity of individual invadopodia. Mechanistically, Orai1 blockade inhibited the recycling of MT1-matrix metalloproteinase (MMP) to the plasma membrane and entrapped MT1-MMP in the endocytic compartment to inhibit ECM degradation. STIM1 knockdown significantly inhibited melanoma lung metastasis in a xenograft mouse model, implicating the importance of this pathway in metastatic dissemination. Our findings provide a novel mechanism for Ca(2+)-mediated cancer cell invasion and shed new light on the spatiotemporal organization of store-operated Ca(2+) signals during melanoma invasion and metastasis.

MicroRNA-182 downregulates metastasis suppressor 1 and contributes to metastasis of hepatocellular carcinoma.

BACKGROUND: miR-182 is one of the most significantly up-regulated miRNAs in hepatocellular carcinoma (HCC). Metastasis suppressor 1 (MTSS1), one target gene of miR-182, plays an important role in the metastasis of cancers. However, it remains unclear what role does function and mechanism of miR-182 and MTSS1play in HCC. METHODS: miR-182 expression was tested in 86 cases of paired HCC and normal tissues by real-time PCR and the relationships between miR-182 expression and clinicopathological parameters were analyzed. The expression of MTSS1 was evaluated by immunohistochemistry and western blot in the above tissues and its correlation with miR-182 expression was analyzed. Moreover, western blot and invasion assays were performed after transfection of pre-miR-182 or anti-miR-182 to HCC cell lines. In addition, luciferase assays was performed to confirm the regulation of miR-182 on MTSS1. RESULTS: Compared with normal tissue, miR-182 was up-regulated and MTSS1 was down-regulated in HCC tissues. Moreover, the over-expression of miR-182 was correlated with intrahepatic metastasis (p = 0.034) and poor prognosis (p = 0.039) of HCC patients. There was a negative correlation between miR-182 and MTSS1 expression in both HCC tissues (r = -0.673, p < 0.01) and HCC cell lines (r = -0.931, p = 0.021). Furthermore, the up-regulation of miR-182 resulted in the down-regulation of MTSS1 and increased invasive potential of HUH-1, and reverse results were also confirmed when the expression of miR-182 was inhibited. In addition, the results of the luciferase assay demonstrated the targeted regulation of miR-182 on MTSS1. CONCLUSIONS: miR-182 could promote metastasis of HCC and inhibit the expression of MTSS1. miR-182 and MTSS1 are potential prognostic markers and/or therapeutic targets in HCC.

Study of embryotoxicity of Fusarium mycotoxin butenolide using a whole rat embryo culture model.

Butenolide, a mycotoxin elaborated by several toxigenic Fusarium species, has been implicated as an etiological factor of Kashin-Beck disease and it is always detected in food from endemic Kashin-Beck disease areas. Although butenolide is considered as a potential health risk to humans and animals, its toxicity targets and mechanism of action have not been fully understood and the knowledge of its developmental toxicity is absent. The present study investigated butenolide embryotoxicity via an in vitro whole embryo culture system using rat embryos. Embryos exposed to butenolide at a concentration of 0.625 mg/L showed and differentiation similar to that of the control embryos (=no observed adverse effect concentration; NOAECwec). The embryonic growth and differentiation were affected, represented as reduced crown-rump length and head length, and decreased number of somites from 1.25 mg/L. Total morphological scores decreased significantly at the concentration of butenolide of 2.5 mg/L. All embryos were malformed at 3.75 mg/L and above (=ICMaxWEC), presenting growth retardation with flexion failure and irregular somite differentiation. The IC503T3 of butenolide as calculated from the balb/c 3T3 cytotoxicity test is 6.45 mg/L. Our study shows that butenolide exerts detrimental effects on embryo development in vitro by inducing growth retardation and differentiation inhibition, and the embryotoxicity effect of butenolide should be treated with caution.

Fascin protein is critical for transforming growth factor ß protein-induced invasion and filopodia formation in spindle-shaped tumor cells.

Fascin, an actin-bundling protein overexpressed in all carcinomas, has been associated with poor prognosis, shorter survival, and more metastatic diseases. It is believed that fascin facilitates tumor metastasis by promoting the formation of invasive membrane protrusions. However, the mechanisms by which fascin is overexpressed in tumors are not clear. TGFß is a cytokine secreted by tumor and mesenchymal cells and promotes metastasis in many late stage tumors. The pro-metastasis mechanisms of TGFß remain to be fully elucidated. Here we demonstrated that TGFß induced fascin expression in spindle-shaped tumor cells through the canonical Smad-dependent pathway. Fascin was critical for TGFß-promoted filopodia formation, migration, and invasion in spindle tumor cells. More importantly, fascin expression significantly correlates with TGFß1 and TGFß receptor I levels in a cohort of primary breast tumor samples. Our results indicate that elevated TGFß level in the tumor microenvironment may be responsible for fascin overexpression in some of the metastatic tumors. Our data also suggest that fascin could play a central role in TGFß-promoted tumor metastasis.