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Published in Oncotarget V8I62, Dec 1, 2017
Metformin alleviates nickel-induced autophagy and apoptosis via inhibition of hexokinase-2, activating lipocalin-2, in human bronchial epithelial cells
Oncotarget. 2017; 8:105536-105552. https://doi.org/10.18632/oncotarget.22317
Yu-Ting Kang, Wen-Cheng Hsu, Chih-Hsien Wu, I-Lun Hsin, Pei-Ru Wu, Kun-Tu Yeh _ and Jiunn-Liang Ko
Yu-Ting Kang1,*, Wen-Cheng Hsu2,*, Chih-Hsien Wu1,3, I-Lun Hsin1,4, Pei-Ru Wu6, Kun-Tu Yeh5,7 and Jiunn-Liang Ko1,8
1Institute of Medicine, Chung-Shan Medical University, Taichung, Taiwan
2Department of Endocrinology and Metabolism, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan
3Basic Medical Education Center, Central Taiwan University of Science and Technology, Taichung, Taiwan
4Inflammation Research & Drug Development Center, Changhua Christian Hospital, Changhua, Taiwan
5Department of Surgical Pathology, Changhua Christian Hospital, Changhua, Taiwan
6Department of Pathology, Cheng Ching Hospital, Taichung, Taiwan
7School of Medicine, Chung Shan Medical University, Taichung, Taiwan
8Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan
*These authors have contributed equally to this work
Kun-Tu Yeh, email: [email protected]
Jiunn-Liang Ko, email: [email protected]
Keywords: nickel; autophagy; hexokinase 2; lipocalin 2; metformin
Received: April 14, 2017 Accepted: June 29, 2017 Published: November 06, 2017
Autophagy is an intracellular recycling and degradation process for regulating tumor progression, survival and drug resistance. Nickel compounds have been identified as human carcinogens. However, the role of nickel-induced autophagy in lung carcinogenesis has not yet been fully elucidated. In this study, we determined that hexokinase 2 (HK2), which phosphorylates glucose and regulates autophagy, is the key mediator in nickel-induced autophagy in lung bronchial epithelial cells. We attempted to investigate the effects of the antidiabetic drug metformin on HK2 expression and lung cancer chemoprevention. Our results showed that metformin decreases nickel-induced autophagy and activation of apoptosis through inhibition of HK2 gene, protein and activity. Furthermore, we demonstrated that lipocalin 2 (LCN2), which is released by neutrophils at sites of infection and inflammation is involved in HK2-driven autophagy pathway. Knockdown of endogenous HK2 and LCN2 by shRNA reduced nickel-elicited autophagy and apoptosis, illustrating that metabolic alteration and inflammatory action are important in nickel-elicited carcinogenesis. We also determined the association between nickel-induced autophagy and apoptosis. Inhibition of nickel-induced autophagy abolished apoptotic cell death in chloroquine-treated, shLC3 Beas-2B cells and Atg5−/− MFFs. From TGCA database and immunohistochemistry analysis, HK2 and LCN2 expression increased in lung squamous cell carcinoma and their related adjacent normal tissues. Taken together, our results demonstrated that metformin alleviates NiCl2-induced autophagy and apoptosis via HK2-driven LCN2 activation in human bronchial epithelial cells. This novel mechanism provides a strategy for targeting nickel-elicited lung cancer progression, as well as for preventing HK2 cumulative damage triggered by environmental carcinogens.
Autophagy is a highly conserved self-degradative process that packages dysfunctional proteins and organelles into cytoplasmic double-membrane vesicles called autophagosomes. It is activated in response to multiple stimuli in cancer progression, providing a source of nutrients and energy for tumor development during starvation, hypoxia and immune response [1, 2]. Nickel is widely distributed, posing occupational and environmental exposure risks. Nickel (II) compounds have been classified as group I human carcinogens by the International Agency Research in Cancer (IARC) of the World Health Organization (WHO). Human exposure to nickel comes from many sources, such as metal industries, rechargeable batteries, electroplating processes and cigarette smoking [3]. Epidemiological studies have indicated that long-term exposure to nickel compounds is the main reason for raised lung and sinus cancer risks among nickel refinery employees [4, 5]. The molecular carcinogenic mechanisms of nickel toxicity are thought to involve oxidative stress, DNA damage, epigenetic alteration, chronic inflammation and regulation of gene expression [6, 7]. In our previous study, exposure to soluble nickel compound nickel chloride (NiCl2) induced epithelial-mesenchymal transition (EMT) in lung epithelial BEAS-2B cells by reactive oxygen species (ROS) generation [8], exhibiting the carcinogenic potential of NiCl2. Although the mechanisms of nickel-induced carcinogenesis have been discussed in detail, nickel-activated autophagy has yet to be fully elucidated.
Metabolic alteration is frequently accompanied by rapid differentiation of cells and malignant cells to provide energy. There is a propensity to metabolize glucose to lactic acid by aerobic glycolysis even under sufficient oxygen, a phenomenon known as the Warburg effect [9]. The critical regulator in this frequent cancer phenotype is mitochondrial-bound hexokinase (HK). HK catalyzes the first step of glycolysis, responsible for the conversion of glucose into glucose-6-phosphate. It is well known that HK2, which is found in insulin-sensitive tissues, such as skeletal muscle and adipose tissue, is the major bound HK isoform expressed in cancers [10, 11]. High level of HK2 expression is associated with poor overall survival and prognosis in many types of cancers [12, 13]. Previous studies have indicated that HK2 facilitates autophagy in response to glucose deprivation, functioning as a molecular switch from glycolysis to autophagy under glucose starvation [14]. However, the manner in which nickel-elicited HK2 contributes to lung carcinogenesis by activating autophagy has yet to be thoroughly investigated.
Lipocalin 2 (LCN2) is a critical inflammatory mediator that is persistently induced during endotoxemia, reflecting the extent of kidney damage and kidney failure [15]. LCN2 is also associated with the pathogenesis of various diseases and is upregulated in many types of cancers. Moreover, it has recently been implicated in multiple cancer tumorigeneses [16, 17]. LCN2 also enhance excessive cell autophagy during ischemia/reperfusion injury [18]. LCN2 deficiency decreases autophagy and inhibits cell proliferation [19]. LCN2 may be an important regulator of tumorigenesis through autophagy and proliferation. In the present study, we investigate the effects of exposure to nickel compounds on LCN2-mediated autophagy.
Metformin is the first-line prescribed drug of choice in the treatment of type 2 diabetes and metabolic syndrome, and has recently emerged as a potential anticancer agent with unanticipated cancer prevention activity. Several epidemiological and clinical studies have found that patients using metformin have decreased cancer incidences, as well as inhibited cancer survival and proliferation, in comparison with those using other antidiabetic medications [20-22]. Numerous publications show that anti-carcinogenic effects of metformin are raised by chemical carcinogens or ionizing irradiation in animal models [23, 24].
Although it has been established that nickel exposure upregulates HK2 expression, the mechanism of signal integration between nickel-induced HK2 and autophagy in lung tumor progression has not been elucidated. It exhibits anticancer effect of metformin through regulation of glucose metabolism. Consequently, the aim of this study is to investigate the role of metformin in diminishing NiCl2-induced HK2, and associated autophagy and cytotoxicity, in lung bronchial cells. This is the first report of HK2-driven inflammatory cytokine LCN2 expression in the promotion of autophagy under nickel exposure. We also clarify the relationship between NiCl2-elicted autophagy and apoptosis, as well as demonstrate the efficiency of metformin in prevention and therapy, following environmental carcinogen exposure.
Nickel induces autophagy via HK2 and LCN2 induction in lung cells
To assess the effects of nickel on cell fate, we evaluated autophagy induction and the generation of relevant proteins in the presence of nickel. Autophagy plays an essential role in lung oncogenesis. At the beginning of autophagy, the cytosolic form of LC3B (LC3B-I, 18 kDa) is converted to the phagophore and autophagosome bound form of LC3B (LC3B-II, 16 kDa). Treatment with various concentrations of nickel for various time periods resulted in cell autophagy in BEAS-2B cells. On western blot, 0.25 mM concentration of nickel significantly induced LC3B-II/LC3B-I ratio after 48 h in BEAS-2B cells (Figure 1A and 1B). In a previous study, nickel accumulation increased cellular glycolytic activity, which is the foremost alteration of energy metabolism in tumorigenesis (the Warburg effect) [25, 26]. In the present study, NiCl2 affected multiple genes in the glycolysis pathway on Agilent SurePrint G3 Human V2 GE 8×60K microarray analysis of KEGG pathway. In particular, treatment with NiCl2 induced a 38-fold increase in HK2 mRNA level. (Supplementary Materials, Supplementary Table 1 and Supplementary Figure 1). Nickel stimulated HK2 and inflammation protein LCN2 expressions in BEAS-2B cells, as demonstrated on western blot (Figure 1A and 1B). To assess the mRNA levels of HK2 in NiCl2-treated cells, BEAS-2B cells and WI-38 fibroblasts were treated with various concentrations of NiCl2 for 48 h and analyzed on RT-PCR and Q-PCR (Figure 1C). To confirm autophagic flux in NiCl2-treated cells, AVO development was detected by staining of late autophagic vacuoles with acridine orange dye. As shown in Figure 1D upper, nickel prompted AVO formation in BEAS-2B cells. To calculate the AVO fractional volume after NiCl2 treatment, flow cytometric analysis was performed. The data indicated that NiCl2 stimulates AVO development in a dose-dependent manner in BEAS-2B cells (Figure 1D lower). Secreted LCN2 levels increased following 0.25 mM NiCl2 treatment in BEAS-2B cells (Figure 1E). These results implied that HK2 and LCN2 are activated in the presence of NiCl2.
Figure 1: NiCl2 induces autophagy and up-regulates HK2 and LCN2 in human bronchial epithelial cells. BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 in (A) dose- and (B) time-dependent manner. The protein levels of LCN2, HK2 and LC3B-II were measured on western blot. β-actin was used as an internal control. The relative ratios of LCN2/ β-actin, HK2/ β-actin and LC3B-II/ LC3-I are shown. (C) BEAS-2B (1×106 cells/ 6 cm dish) and WI-38 (5×105 cells/ 6 cm dish) were treated with various doses of NiCl2 for 48 h. The mRNA level of HK2 was measured on RT-PCR and real-time PCR. Quantification of HK2 by qPCR was normalized to the level of GAPDH, with the average of three independent readings. (D) Acridine orange (1μg/mL) was used to stain AVOs in NiCl2 (0, 0.06, 0.12, 0.25 mM)-treated BEAS-2B cells (2×105 cells/ 12-well plate) for 48 h. The fluorescence-activated cells were visualized under a red filter fluorescence microscope and quantified by flow cytometry. Scale bar, 20 μm. (E) NiCl2 increased the secretion of LCN2 in BEAS-2B cell culture supernatant as revealed on ELISA. The data are expressed as mean±SD. *p<0.05, ***p<0.001 on two-tailed t test compared with control.
Metformin represses autophagy and apoptosis by inhibition of NiCl2-induced HK2 levels and activity
HK catalyzes the first step of glycolysis, phosphorylating glucose to glucose-6-phosphate. HK2 plays an important role not only in glycolysis, but also in cell survival. We investigated whether HK2 is involved in NiCl2-induced cell fate. NiCl2 increased HK2 mRNA levels and metformin diminished NiCl2-induced HK2 expression in a dose dependent manner (Figure 2A upper). The same results were obtained on real time RT-PCR for the detection of mRNA expression of HK2 in BEAS-2B cells (Figure 2A lower). As shown in Figure 2B, we performed western blotting to examine whether metformin diminishes up-regulation of the protein levels of HK2 in the presence of NiCl2. To quantify the HK2 protein expression affected by NiCl2 and metformin on western blotting, we repeated the same experiment three times. As 2-DG is an analog of glucose, it has been used as an HK inhibitor, as it competes with glucose. Remarkably, 2-DG treatment also decreased NiCl2-induced HK2 expression (Figure 2C). As shown in Figure 2D, metformin and 2-DG significantly decreased NiCl2-induced HK activity. Moreover, the decrease in HK2 expression was based on two different short hairpin RNAs (shRNAs). The responses of BEAS-2B shGFP cells were similar to those of parental BEAS-2B cells after NiCl2 and metformin treatment. In BEAS-2B shHK2 cells, NiCl2-elicited LC3B-II and cleaved caspase-7 expressions were significantly diminished. In addition, there was strong correlation between the expression of HK2 and LCN2 level. Moreover, BEAS-2B shHK2 cells significantly decreased following NiCl2 treatment (Figure 2E). To confirm the effect of HK2 silencing on AVO fraction volume after NiCl2 and metformin treatment, flow cytometry was performed (20.6% versus 7.1% and 10.4%) (Figure 2F). These data indicated that HK2 is involved in the induction of autophagy in the presence of NiCl2. It is well known that the generation of reactive oxygen species (ROS) contributes to nickel-triggered carcinogenesis, including EMT promotion and the cause of DNA damage [8, 27]. To determine whether metformin can suppress NiCl2-induced ROS accumulation, cells were treated with 2′, 7′ -dichlorodihydrofluorescein diacetate (H2DCFDA) and analyzed by flow cytometry. Results revealed that metformin decrease ROS generation in the presence of nickel (10.42% versus 5.58%). N-acetyl-cysteine (NAC, 1 mM), the ROS scavenger, was used to confirm the reversion of NiCl2-induced ROS (Figure 2G).
Figure 2: Inhibition of NiCl2-induced hexokinase 2 represses autophagy and apoptosis. (A) BEAS-2B cells (1×106 cells/6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) for 48 h. The mRNA levels were measured on RT-PCR and real-time PCR. *p<0.05, **p<0.01, ***p<0.001, two-tailed t test. (B, C) BEAS-2B cells (1×106 cells/6 cm dish) were co-treated with 0.25 mM NiCl2, metformin (0, 1, 2.5, 5 mM) or 2-DG (0, 1, 2.5, 5 mM) for 48 h. The protein levels were determined on western blot analysis. β-actin was used as the internal control. Statistical analysis of western blotting. The protein levels of HK2 were standardized by β-actin protein level. *p<0.05, **p<0.01, ***p<0.001, two-tailed t test. (D) Quantification of HK activity from whole-cell lysates showed a significant decrease following treatment with 0.25 mM NiCl2 combined with 5 mM metformin or 5 mM 2-DG for 48 h. The level of HK activity was determined by OD 450. ***p<0.001, two-tailed t test. (E) After 0.25 mM NiCl2 and 5 mM metformin treatment, equal amounts of total lysates from BEAS-2B shGFP and shHK2 cells (1×106 cells/6 cm dish) were analyzed on western blot. The relative ratios of HK2/β-actin, LC3-II/LC3-I and cleaved caspase 7/β-actin are shown. (F) Flow cytometric analysis of the NiCl2- and metformin-treated cells after staining with acridine orange for the quantification of AVOs. (G) BEAS-2B cells were pretreated with 10 mM NAC for 1 h followed by exposure to 0.25 mM NiCl2 and 5 mM metformin for 48 h. The intracellular ROS levels of the cells were measured by flow cytometry analysis staining with H2DCFDA.
Endogenous LCN2, but not exogenous LCN2, triggers NiCl2-mediated autophagy in bronchial epithelial cells
LCN2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is required for tumor progression and metastasis. It is often implicated in the responses to hypoxia and apoptosis induction [28]. However, the correlation between LCN2 and autophagy in the presence of NiCl2 remains unclear. Actually, a causal link between LCN2 and HK2 levels and autophagy levels in bronchial epithelial cells has not been reported, which prompted us to clarify whether LCN2 is involved in NiCl2-elicited autophagy. To assess the effect of metformin on NiCl2-induced LCN2 expression, BEAS-2B cells were cultured in the presence of NiCl2 with or without metformin for 48 h, and RT-PCR and qPCR were performed to detect the mRNA expressions of LCN2. As shown in Figure 3A, metformin significantly decreased NiCl2-induced LCN2. In addition, treatment with metformin or 2-DG reduced NiCl2-elicited LCN2 protein levels. The same results were obtained on ELISA for secretion of LCN2 in a BEAS-2B cell culture supernatant following NiCl2, metformin or 2-DG treatment (Figure 3B, 3C). Particularly, the protein level of LCN2 was down-regulated approximately 40% in all cell lysates following 2-DG treatment. However, in metformin-treated cells, NiCl2–induced secretion of LCN2 was blocked (Figure 3B, 3C). These results revealed that NiCl2–mediated LCN2 is repressed by metformin at translational and transcriptional levels.
Figure 3: LCN2 is involved in NiCl2-induced autophagy in human bronchial epithelial cells. (A) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) for 48 h. The mRNA level of LCN2 was determined on RT-PCR and qPCR. Quantification of LCN2 by qPCR was normalized to the level of GAPDH, with the average of three independent readings. The data are expressed as mean ± S.D. *p<0.05, ***p<0.001 on two-tailed t test. (B) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) or 2-DG (0, 5 mM) for 48 h and subjected to western blotting for LCN2. (C) Metformin or 2-DG decreased NiCl2-induced secretion of LCN2 in BEAS-2B (2×105 cells/12-well plate) cell culture supernatants, as revealed on ELISA. The data shown are the mean ± S.D. of three independent experiments. ***p<0.001 compared to control and ###p<0.001 compared to Ni-treated cells, two-tailed t test. (D) Cells (1×106 cells/6 cm dish) were treated with LCN2 (10 ng/mL) for 0, 6, 12, 24, 48 h. HK2, LC3B levels were analyzed on western blot. β-actin served as the loading control. (E) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with 0, 0.4, 2, 10, 40 ng/mL LCN2 for 48 h. The cells were stained with acridine orange (1 μg/mL) for AVO observation and analyzed using flow cytometry. (F) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 and metformin for 48 h after infection with lentivirus carrying shLCN2 #89, shLCN2 #90 or vector control (shGFP). The protein levels of LCN2, HK2, LC3B were detected on western blot. β-actin was used as an internal control. (G) Quantitative detection of AVOs by acridine orange staining in cells was performed using flow cytometry.
We further examined whether LCN2 independently prompts autophagy and attempted to clarify the relationship between LCN2 and HK2 in lung epithelial cells. Western blot analysis was performed to observe whether LCN2 treatment induces the processing of LC3B-I to LC3B-II, and the protein expression of HK2. The results demonstrated that HK2 and LC3B-II/I do not accumulate following LCN2 treatment (10 ng/mL) for up to 48 h (Figure 3D). We utilized acridine orange staining to confirm this result. Concentrations of LCN2, up to 40 ng/mL, failed to alter the increase in AVO accumulation (Figure 3E). Furthermore, the protein levels of HK2 and LC3B-II/I ratios were determined after attenuation of LCN2 activity by small hairpin RNA (shRNA). The data revealed that blockade of endogenous LCN2, but not exogenous LCN2, represses autophagy in BEAS-2B cells (Figure 3F). To calculate the AVO fractional volume, we performed flow cytometric analysis 48 h after LCN2 silencing and co-treatment with NiCl2 and metformin (Figure 3G). Taken together, the results suggested that LCN2 is involved in NiCl2-induced autophagy.
NiCl2-mediated autophagy decreases following metformin treatment in human bronchial epithelial cells
To determine the intracellular distribution of ionic nickel in the cells, we utilized Ni2+-selective fluorescence dye Newport GreenTM DCF, which fluoresces when there is binding with ionic nickel ions. As shown in Figure 4A upper, in a dose-dependent experiment, BEAS-2B cells were exposed to various concentrations of NiCl2 for 48 h. The result was an incremental increase in green fluorescence in NiCl2-treated cells. In a previous study, cellular responses of metformin were associated with this drug's metal-binding properties, especially binding with copper. However, the association with nickel remained unclear [29]. To further investigate whether metformin sequesters intracellular nickel, BEAS-2B cells were co-treated with 0.25 mM NiCl2 and 5 mM metformin for 48 h, then stained with Newport GreenTM DCF. As shown in Figure 4A lower, treatment with metformin did not affect NiCl2-activated fluorescence, indicating that metformin does not contribute to blockage of nickel ions into cells. To evaluate the effect of metformin on NiCl2-elicited autophagy, we utilized pEGFP-LC3 transient transfection to visualize aggregation of expression of LC3B. After treatment with 0.25 mM NiCl2, GFP-LC3 was redistributed from a ubiquitous, diffuse pattern toward autophagosomes, observed as cytoplasmic dots in BEAS-2B cells. Remarkably, treatment with 5 mM metformin decreased the formation of GFP-LC3 puncta (Figure 4B). We then examined the effect of metformin on NiCl2-induced AVO development. Treatment with metformin blunted AVO formation in a dose-dependent manner in NiCl2-treated cells. Flow cytometric analysis was performed to quantify the AVO fractional volume (18.35% versus 6.98%) (Figure 4C).
Figure 4: Effects of metformin on NiCl2-mediated autophagy in human bronchial epithelial cells. (A) BEAS-2B cells (2×105 cells/well of 12-well plate) were treated with varying doses of NiCl2 (0, 0.06, 0.125, 0.25 mM) (top), or with NiCl2 (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM), for 48 h (bottom). The cells were stained with Newport Green™ DCF diacetate (1 μM) for green fluorescence observation under a green filter fluorescence microscope. Scale bar, 20 μm. (B) pEGFP-LC3B transfection revealed LC3B puncta (green) in BEAS-2B cells (8×104 cells/well of 24-well plate) treated with NiCl2 (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) for 48 h. Cells were fixed and stained with DAPI for nuclear visualization under a confocal microscope. Dashed lines encircle the enlarge images and arrow heads point to the puncta of GFP-LC3. Scale bars, 20 μm. (C) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) for 48 h and stained with acridine orange (1 μg/mL) for AVO observation. Cells were visualized under a red filter fluorescence microscope with quantification by flow cytometry. (D) Western blots of p-AKT, p-P70S6K, p-AMPK, LC3B and (E) autophagy-related genes Beclin-1, Atg12-Atg5 and Atg3 expressions in protein lysates from BEAS-2B cells (1×106 cells/ 6 cm dish) treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) for 48 h. β-actin was used as an internal control. The relative ratios of p-AKT/β-actin, p-P70S6K/β-actin, p-AMPK/β-actin, LC3B-II/β-actin, Beclin-1/β-actin, Atg12-Atg5/β-actin and Atg3/β-actin are shown. (F) p-AMPK and p-P70S6K were determined by western blotting after BEAS-2B shGFP and shHK2 cells (1×106 cells/ 6 cm dish) were treated with NiCl2 and metformin for 48 h. β-actin was used as an internal control.
mTOR, the Akt and AMPK downstream effector, plays a critical role in cell proliferation, growth and survival. Activated mTOR promotes protein translation by phosphorylating its substrates, including p70 ribosomal protein S6 kinase (p70S6K) [30]. To identify the effects of metformin on NiCl2-induced autophagy-related pathway and genes, NiCl2 and metformin were administered for 48 h, followed by analysis of protein expressions on western blotting. As shown in Figure 4D and Figure 4E, Akt-Ser473 phosphorylated level was downregulated, but did not affect phosphorylated p70S6K-Thr389, AMPK-Thr172, beclin-1, ATG12-ATG5 and ATG3 levels after 0.25 mM NiCl2 treatment for 48 h. Beclin-1, ATG12-ATG5 and ATG3 are members of the ATG family involved in the formation of autophagosomes [31]. Metformin treatment revived the Akt-Ser473 phosphorylated expression, but reduced NiCl2-mediated autophagy, perhaps through Akt-dependent but mTOR-independent pathway. In addition, metformin is known to activate AMPK. To determine the relationship between HK2 and p-AMPK on nickel-induced autophagy, shGFP and shHK2 BEAS-2B cells treated with NiCl2 and metformin were performed. As shown in Figure 4F, there were no significant changes in the protein levels of p-AMPK and p-P70S6K after knockdown of HK2.
NiCl2-elicted autophagy contributes to activation of apoptosis
It is well documented that nickel exposure induces apoptosis via ROS accumulation and involvement of mitochondria, ER-stress, Fas, and c-Myc [32]. To investigate the effects of metformin on NiCl2-mediated apoptosis and to clarify the cellular sources of HK2 in autophagy and apoptosis in NiCl2-treated cells, BEAS-2B cells were treated with 1, 2.5 or 5 mM metformin, with or without 0.25 mM NiCl2, for 48 h and analyzed by flow cytometry. As shown in Figure 5A, NiCl2-mediated apoptosis was recovered by metformin (13.22% versus 3.17%). In addition, we assessed the protein markers of apoptosis and autophagy in the presence of NiCl2, with or without metformin or 2-DG, on western blotting. Metformin or 2-DG treatment simultaneously abolished the increases in LC3-I/LC3-II ratio and cleaved caspase-7 expression in NiCl2-treated BEAS-2B cells (Figure 5B). To further determine whether NiCl2-mediated autophagy stimulates apoptosis, the autophagy inhibitor chloroquine (CQ) was used to suppress late phase autophagy. As shown in Figure 5C, treatment with NiCl2 and metformin, as well as with 10 μm CQ, blocked the endogenous LC3-II turnover and resulted in increased NiCl2-elicted autophagy. CQ treatment also inhibited NiCl2-mediated cleavage of poly ADP-ribose polymerase and cleavage of caspase-7, which served as apoptotic markers. Particularly, metformin prevented the accumulation of LC3-II and apoptotic proteins, with or without CQ treatment. To further confirm the correlation between autophagy and apoptosis in cells exposed to NiCl2, we observed atg5 knockout mouse embryonic fibroblast cells (Atg5-/- MEF cells), as well as LC3 knocked-down BEAS-2B cells. In atg5-/- and atg5 wild-type MEF cells, NiCl2 treatment slightly increased the protein expression of HK2. Similar to NiCl2-treated BEAS-2B cells, in atg5 WT MEFs there was significant induction of LC3 I to II conversion, as well as cleavage of PARP and caspase 3 expression. However, there was failure to prompt atg5-/- MEFs (Figure 5D). As shown in Figure 5E, the specific shRNA targeting LC3 was transfected into BEAS-2B cells with knockdown of the expression of LC3. In comparison with BEAS-2B shGFP cells, NiCl2-induced cleavage of caspase 7 was blunted in BEAS-2B shLC3 cells. Our results demonstrated that NiCl2-induced autophagy induces apoptosis.
Figure 5: Inhibition of autophagy decreases NiCl2-elicted apoptosis. (A) Flow cytometry analysis of BEAS-2B cells (1×106 cells/ 6 cm dish) treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) for 48 h, after which cells were stained with Annexin V/PI. (B) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) or 2-DG (0, 5 mM) for 48 h and subjected to western blotting for HK2, cleaved caspase 7 and LC3B. β-actin was used as an internal control. The relative ratios of HK2/β-actin, cleaved caspase 7/β-actin and LC3B-II/LC3-I are shown. (C) Western blot of BEAS-2B cells (1×106 cells/6 cm dish) treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) with or without CQ (0, 10 μM) for 48 h. (D) Atg5 wild type (WT) and Atg5-/- MEF cells (2×105 cells/6 cm dish) were treated with NiCl2 (0, 0.25 mM) for 48 h. β-actin was used as an internal control. The relative ratios of HK2/β-actin, LC3B-II/LC3B-I, cleaved PARP/β-actin and cleaved caspase 3/β-actin are shown. (E) Cleaved caspase 7 and conversions of LC3-I to LC3-II were determined by western blotting after BEAS-2BshLuc and shLC3 cells (1×106 cells/ 6 cm dish) were treated with NiCl2 and metformin for 48 h. β-actin was used as an internal control.
HK2 is the crucial regulator in lung cancer progression
We assessed HK2 and LCN2 expressions in The Cancer Genome Atlas (TCGA) Data Portal from Broad GDAC Firehose and performed immunohistochemical staining to detect the expressions of HK2 and LC3B in 72 human lung cancer specimens to determine whether HK2, LCN2 and LC3B are involved in lung cancer progression. The representative IHC results are shown in Figure 6A. The presence or absence of HK2 and LC3B protein expressions was associated with tumor stage, T status and metastasis (Supplementary Table 2). Both HK2 and LCN2 expressions significantly increased in cancer tissues when compared with normal tissues in lung squamous cell carcinoma (Figure 6B). Furthermore, we examined the expressions of HK2 and LCN2 in LUSC and LUAD tissues and their corresponding noncancerous tissues using the TCGA Data Portal (Figure 6C). The results revealed that HK2 and LCN2 are associated with tumor progression, especially in LUSC tissue.
Figure 6: HK2 and LCN2 levels increased in lung squamous cell carcinoma and lung adenocarcinoma samples. (A) IHC HK2 protein expressions in NSCLC patients. (Left) No tumor tissue. (Middle) Low expression immunostaining. (Right) High expression immunostaining. Scale bars, 200 μm. (B) The expressions of HK2 and LCN2 in normal and lung squamous cell carcinoma and adenocarcinoma from TCGA Data Portal. (C) Relative expressions of HK2 and LCN2 in 50 or 57 pairs of lung squamous cell carcinoma or adenocarcinoma tumor tissues and their corresponding adjacent non-cancerous tissues.
It is well documented that metformin alleviates autophagy and apoptosis via HK2 and LCN2, following exposure to nickel, in bronchial epithelial cells. The molecular basis of nickel carcinogenicity has proven complex, as many chronic inflammation and stress response pathways are activated in nickel-specific toxicology profiles. There is much evidence that chronic inflammation contributes to the onset and progression of cancer [33, 34]. In recent studies, Toll-like receptor 4 (TLR4) has been identified as the critical mediator of the innate immune response to nickel that triggers NF-κB signaling and pro-inflammatory gene activation [35]. Although nickel compounds have low mutagenic capabilities, previous studies have found that nickel accumulation in lung tissues contributes to incremental levels of EGFR and P53 mutations, which can reduce DNA repair activity and promote tumor invasion, leading to lung carcinogenesis [36-38]. In addition, accumulating evidence has emphasized the importance of nickel in modulating the epigenetic landscape that includes chromatin structural modifications, DNA methylation and histone modifications [39, 40]. Nickel also induces the up-regulation of a specific set of proteins and microRNAs (miRNAs), leading to altered DNA methylation and histone modification landscapes in a variety of cell types [7, 39]. From the results of recent studies, HK2 is highly expressed in various cancers, and is regulated by miRNA [41]. MiR-143, an anti-oncomiR, is often downregulated in cancers, such as colon and gastric cancers, as well as B-cell lymphoma [42-44]. It targets HK2 mRNA and inhibits HK2 expression [41, 45]. Moreover, other miRNAs may be involved in the altered expression of HK2 in tumors, including miR-181b, miR-125b and miR-182 [46-48].
Nickel-induced carcinogenesis may involve glycolysis pathway activation. It has been shown that genes related to glucose metabolism and glycolysis are inducible by nickel exposure in an HIF-dependent manner [49]. Actually, HIF has been found to accumulate in various cell lines in the presence of nickel [50]. Increasing numbers of studies have shown that metabolic enzymes directly contribute to carcinogenesis. In comparison with normal tissues, cancer cells prefer to metabolize glucose into lactic acid by glycolysis, which is known as the "Warburg effect", and is accompanied by upregulation of HK2 [9]. Previous studies have demonstrated that HK2 is highly present in lung and breast cancers, and is required for tumor initiation and maintenance. Tumor progression is impaired following its downregulation [51]. Here, we demonstrated that reduction in NiCl2-induced HK2 by metformin inhibits NiCl2-mediated autophagy. It is known that metformin suppresses hypoxia-induced HIF-1α accumulation [52]. In this study, we confirmed that metformin decreases nickel-induced HIF-1α expression (data not shown). We also demonstrated the importance of HK upregulation in nickel-induced autophagy through inhibition of the expression and activity of HK2 using competitive HK inhibitor 2-DG and HK2-specific shRNA silencing. HK2 silencing combined with metformin demonstrated that autophagy is not only inhibited by HK2 activation (Figure 2E).
A previous study demonstrated that HK2 positively regulates protective autophagy via TORC1 inhibition in response to glucose deprivation [14]. However, in contrast with normal tissues, inhibition of HK2 by 2-DG suppresses lung cancer cell growth through induction of cell apoptosis and autophagy [53]. Furthermore, treatment with 2-DG and CQ represses HK2-mediated Warburg effect and ULK1-dependent autophagy activates apoptosis to cause tumor regression [54]. These studies indicated that HK2 is able to regulate different effects of autophagy and is a key mediator and energy precursor. Therefore, we investigated metformin as a new anti-autophagy drug by targeting Ni-accumulated HK2 in lung epithelial cells.
In our previous study, treatment with NiCl2 stimulated EMT via HIF-1α-dependent pathway and E-cadherin promoter hypermethylation in bronchial epithelial cells [8]. EMT is an important step in the progression of lung cancer toward metastasis and invasion and occurs during the development of epithelial carcinogenesis [55, 56]. Actually, the correlation between EMT and autophagy has been well studied over the past decade. It has been observed that autophagy contributes to cancer invasion through EMT activation during starvation or hypoxia [57-59].
A recent report showed that HK2 overexpression is associated with the hypomethylation status of CpG island region -379 to +209 from HK2 promoter in hepatocellular carcinomas [60]. It had been stated that DNA hypomethylation of proto-oncogene contribute to nickel-induced malignant transformation [61]. We could not rule out the possibility that nickel will regulate the epigenetic alteration of HK2 promoter hypomethylation. In addition, metformin may obstruct HIF-1α binding on HK2 promoter resulting in HK2 decrease. Overall, these results evidence that the potential capacity of nickel alter the epigenetic regulation and the transition to the status of higher aggressiveness and thus progression of carcinogenesis.
It is worth noting that autophagy induced by TLR4 signaling promotes TLR-triggered cytokine production, which accelerates migration and invasion of lung cancer cells [62]. Studies have suggested that LCN2 upregulated by LPS is influenced via TLR4 signaling pathway [63, 64]. In this study, we demonstrated that NiCl2 induces autophagy induction via increment levels of HK2 and LCN2. As shown in Figure 2F and Figure 3F, we used acridine orange stain to verify the role of HK2 and LCN2 in nickel-induced autophagy after HK2 and LCN2 gene silencing. The results demonstrated that NiCl2-elicited autophagy is via HK2-LCN2 pathway. Although we suggested that LCN2 is involved in NiCl2–induced autophagy pathway, we could not clarify whether nickel upregulates LCN2 expression via TLR4 signaling or if nickel-induced autophagy and EMT activation are facilitated by TLR4 at the source. We did find that metformin restores the protein expression of E-cadherin (data not shown). In future studies, we will explore the precise mechanism of metformin in diminishing the various effects of nickel.
Nickel induced malignant transformation through SQSTM1/P62 and inflammatory TNF upregulation in Beas-2B cells [65]. Son et al. found that nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in nickel-induced autophagy [66]. In the present study, we found that nickel induce autophagy via HK2 and LCN2. Autophagy is triggered by the stress of metabolism and inflammation. Our results suggest that autophagy is one of cancer-promoting reasons under nickel exposure. In fact, autophagy has been shown to play a protective role against apoptosis in malignant transformation. Whether autophagy promotes cell survival or cell death depends on the levels of stress. When stress severity or duration increases, cell death may result. In brief, autophagy affects cellular homeostasis [67, 68]. From our data, nickel simultaneously induces autophagy and apoptosis. To further demonstrate the association of autophagy with apoptosis in the presence of nickel, endogenous LC3 was knocked down by shRNA treatment, and WT or Atg5-/- MEF cells were treated with CQ (Figure 5C-5E). We observed that the activation time point of nickel-induced autophagy is earlier than that of apoptosis (data not shown). We used short-term exposure and high concentrations of nickel to clarify the role of nickel-induced autophagy. In the presence of excessive nickel, there is an imbalance in autophagy, which promotes apoptosis.
There are some limitations in the study. Nickel contents were hardly determined in tissue array. We could not analyze the samples for the relationship of nickel exposure and HK2 expression. However, we investigate the tumorigenesis of HK2 and LCN2 on TCGA database (Figure 6B, 6C). Both HK2 and LCN2 serve as biomarkers in lung cancer progression. We also observed the expression of HK2, LCN2 and autophagy-related genes in lung cancer cell lines, including CL1-0, CL1-5, TL-6 and H1975 with or without 0.25 mM NiCl2 (Supplementary Figure 2A, 2B). Results revealed that most of cancer cells elevate the expression of HK2 and LC3B in the presence of nickel. Equivalently with BEAS-2B cells, autophagy-related genes Atg5 and Beclin-1 except LC3B were decreased after NiCl2 treatment.
In conclusion, the results of this study provide evidence that metformin alleviates NiCl2-stimulated autophagy via the inhibition of HK2 and LCN2 expressions (Figure 7). Accumulation of nickel triggers metabolic changes and inflammatory environment contributes to lung cancer development. The results of this study also demonstrated the preventive effects of metformin against cumulative damage caused by environmental carcinogens.
Figure 7: Proposed model and hypothesis of metformin-induced repression of HK2-driven autophagy following nickel exposure in BEAS-2B cells. Nickel-induced HK2 is a potent mediator of autophagy activation in BEAS-2B cells. We demonstrated the therapeutic efficacy of metformin in the prevention of toxicity of environmental carcinogens.
Human lung specimens and cell lines
Human bronchial epithelial cell line BEAS-2B (CRL-9606TM, ATCC) and lung fibroblast cell line WI-38 (CCL-75™, BCRC) were purchased from the American Type Culture Collection and BCRC (Taiwan), respectively. Tissue specimens were collected in tissue microarray (SuperBiochips) obtaining from GenDiscovery. Tumor types and stages were determined according to WHO classification (World Health Organization, 1981) by experienced clinical pathologists. This study was approved by the Chung Shan Medical University Hospital Institutional Review Board (CSMUH IRB CS16101).
Cell culture and chemicals
BEAS-2B cells were routinely grown in serum-free LHC-9 medium (GIBCO, 12680-013). WI-38 cells were cultured in Minimum Essential Medium (MEM) (GIBCO, 11095-080) containing 10% heat-inactivated fetal bovine serum (FBS) (GIBCO, 10437), 100 ng/mL each of penicillin and streptomycin (GIBCO, 15140), 1 mM nonessential amino acid and 1 mM sodium pyruvate. Both types of cells were incubated in humidified atmosphere of 5% CO2 at 37°C. Reagents used in the study included NiCl2 (Sigma, N6136), metformin (Sigma, D150959), 2-deoxy-D-glucose (2-DG, Sigma, D6134), chloroquine (CQ) diphosphate salt (Sigma, C6628), and recombinant human LCN2 (Biolegend, 588102).
Following treatment protocols, cells were washed twice with ice-cold PBS (2.7 mMKCl, 2 mM KH2PO4, 137 mMNaCl, 10 mM Na2HPO4, pH 7.4), then lysed with RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail (Roche, 04693116001). The concentration of protein extracts was assayed with Bio-Rad Protein Assay Kit (Bio-Rad, 500-0006). Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to hydrophobic polyvinylidene fluoride transfer membrane (PVDF) (PALL, 29301-808). Membranes were blocked in TBS containing 5% non-fat milk and 0.2% Tween 20 for 1 h and probed overnight at 4°C with primary antibodies. Antibodies against HK2 (2867), PARP (9542), LC3B (3868), cleaved caspase-7 (9491), cleaved caspase-3 (9661), p-AKTSer473 (9271) and p-AMPKαThr172 (2535) were obtained from Cell Signaling Technology. Antibody against p-p70S6kThr389 (MABS82) was purchased from Millipore. Antibody against LCN2 (AF1757) was supplied by R&D Systems and β-actin (A5441) was supplied by Sigma. Blots were washed in 0.2% TBS-Tween 20 and incubated with HRP-conjugated secondary antibodies for 1 h. The protein blots were visualized using enhanced chemiluminescence reagent obtained from Perkin Elmer (NEL105001EA).
RNA extraction, reverse transcription-PCR and quantitative real time-PCR analysis
Total RNA was extracted from harvested cells using the rare RNA reagent (Genepure Technology) per the manufacturer's instructions. We quantified RNA and reverse transcribed cDNAs from 3 μg of total RNA per 20 μL RT reaction using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813). PCR was carried out with the following primers: HK2 forward: 5′-ATG AGG GGC GGA TGT GTA TCA-3′, reverse: 5′-GGT TCA GTG AGC CCA TGT CAA-3′; LCN2 forward: 5′-GAG TTA CCC TGG ATT AAC GA-3′, reverse: 5′-CTC CTT TAG TTC CGA AGT CA-3′; LC3B forward: 5′-GCC GTC GGA GAA GAC CTT CAA G-3′, reverse: 5′-TGG TGT GGA GAC GCT GAC CAT G-3′; Atg5 forward: 5′-TTT GCA TCA CCT CTG CTT TC-3′, Atg5 reverse: 5′-TAG GCC AAA GGT TTC AGC TT-3′; Beclin-1 forward: 5′-GGC TGA GAG ACT GGA TCA GG -3′, Beclin-1 reverse: 5′-CTG CGT CTG GGC ATA ACG-3′; β-actin forward: 5′-TCA TCA CCA TTG GCA ATG AG-3′, reverse: 5′-CAC TGT GTT GGC GTA CAG GT-3′. PCR products were separated onto 1.5% agarose gel and visualized by ethidium bromide staining. Real time-PCR was performed using ABI StepOnePlus real time PCR system with gene-specific primers and Smart Quant Green Master Mix with dUTP & ROX (Protech, PT-GL-SQGLR-V3). The mRNA of β-actin was used to standardize the total amount of cDNA on real-time PCR.
Human LCN2 enzyme-linked immunosorbent assay (ELISA)
ELISA was performed using Human LCN2/NGAL DuoSet ELISA Kit (R&D Systems, DY1757) according to the manufacturer's instructions. The absorbance at 450 nm was measured using a microplate reader.
Detection and quantification of acidic vesicular organelles with acridine orange
Autophagy is the process of packaging cytoplasmic proteins into the lytic component and characterized by the formation of acidic vesicular organelles (AVOs). After treatment for 48 h, cells were washed in PBS and stained with acridine orange (Sigma, A6014) (1 μg/ml) in serum-free LHC-9 medium for a period of 15 min, then washed twice with PBS and suspended in LHC-9. To observe the formation of AVOs, the cells were detected under a red filter fluorescence microscope and quantified using flow cytometry.
HK activity assay
HK was assayed according to the manufacturer's instructions using HK Colorimetric Assay Kit (Biovision, K789-100). To assay total HK activity after treatment with NiCl2 and metformin, cell lysates were homogenized with ice cold HK Assay Buffer and the supernatant was collected with reaction mixture. This was followed by incubation for 5 min at room temperature and measurement at excitation wavelength of 450 nm for 30 min. Specific activity was determined using NADH standard. All experiments were repeated at least three times.
VZV-G pseudotyped lentivirus-shRNA system
RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica. Individual clones were identified by their unique TRC number: shGFP TRCN0000072178 (responding sequence: CAA CAG CCA CAA CGT CTA TAT) and shLuc TRCN0000072246 (responding sequence: CAA ATC ACA GAA TCG TCG TAT) for vector control; shHK2 (27) TRCN0000232927 (responding sequence: TGA CGA CAG CATC ATT GTT AA) and shHK2 (82) TRCN0000195582 (responding sequence: CCA AAG ACA TCT CAG ACA TTG) targeted to HK2; shLCN2 (89) TRCN0000060289 (responding sequence: CCA GCA TGC TAT GGT GTT CTT) and shLCN2 (90) TRCN0000060290 (responding sequence: GTA CTT CAA GAT CAC CCT CTA) targeted to LCN2; shLC3 (87) TRCN0000243387 (responding sequence: GGT GAT CAT CGA GCG CTA CAA) and shLC3 (91) TRCN0000243391 (responding sequence: AGC GAG TTG GTC AAG ATC ATC) targeted to LC3. The cells were selected with 2 μg/ml puromycin (Sigma, P8833).
Detection of intracellular nickel
Intracellular mobilized ionic nickel was detected after NiCl2 and metformin treatment using green fluorescent Newport GreenTM DCF diacetate indicator dye (Invitrogen, N7991). The treated cells were washed twice with HBSS and incubated with 1 μM Newport GreenTM DCF diacetate in LHC-9 for 30 minutes. Then, they were washed twice with HBSS after recovery in LHC-9 containing Newport GreenTM DCF diacetate. Green fluorescence was visualized under an Olympus CK40 fluorescence microscope.
pEGFP-LC3 plasmid transfection
pEGFP-LC3 expression vector was purchased from Addgene (#21073). The pEGFP-LC3 plasmid is a pEGFP-C1 plasmid inserted into microtubule-associated protein 1 light chain 3 (LC3) cDNA at the C-terminus and green fluorescent protein (GFP) at the N-terminus [69]. pEGFP-LC3 fusion protein was used to visualize the autophagosomes in cells. Transfection was performed on 24-well plates with coverslips, with 1 μg plasmid in each well and jetPEI transfection reagent (Polyplus-transfection, 101-10). This was followed by incubation overnight. The medium was removed and fresh medium containing NiCl2 and metformin was added to the wells for 48 h. After exposure, the cells were washed twice with PBS and fixed in 3.7% paraformaldehyde-PBS for 10 min at room temperature. Observation of GFP-LC3 puncta in cells was carried out under confocal microscope (ZEISS LSM510 META).
Detection of intracellular reactive oxygen species (ROS)
Cellular ROS was detected using the fluorescence probe 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen, D399). After nickel and metformin treatment for 48 h, BEAS-2B cells were stained with 20 μM H2DCFDA at 37 °C for 30 min in the dark. Then washed twice with PBS and harvested cell in PBS contained with 5% FBS. The fluorescence intensity was analyzed using flow cytometry.
Expression analysis of the cancer genome atlas lung squamous cell carcinoma and lung adenocarcinoma data
Gene expression data were obtained from The Cancer Genome Atlas (TCGA) lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) datasets (https://tcga-data.nci.nih.gov/tcga). The datasets contain data from 463 LUSC samples with 50 adjacent normal tissue samples and 488 LUAD samples with 57 adjacent normal tissue samples, respectively.
Antibodies against HK2 (2867) and LC3B (3868) were obtained from Cell Signaling Technology. Negative controls were used, leaving out the primary antibody. Immunohistochemical methods were carried out using conventional streptavidin peroxidase method according to the manufacturer's (Dako) LSAB Kit (K675) procedure. Slides were visualized using 3,3'-diamino-benzidine tetrahydrochloride as a substrate. The control slide (Dako, T1076) and semi-quantitative H scores of HK2 immunoreactivity were determined by multiplying the proportional scores of stained cells by their immunoreactivity intensity. All immunohistochemical staining cases were examined by two pathologists (Pei-Ru Wu and Kun-Tu Yeh, Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan), and a final agreement was obtained for each score at a discussion microscope.
Statistical analyses were performed using SPSS statistical software (version 18.0; SPSS, Inc., Chicago, IL). All statistical tests were two-sided, and a p value of less than 0.05 was considered statistically significant. Values presented are the means ± standard deviation (SD) of at least three independent experiments.
HK2, hexokinase 2; LCN2, lipocalin 2; NiCl2, nickel chloride; EMT, epithelial-mesenchymal transition; ROS, reactive oxygen species; KEGG, Kyoto Encyclopedia of Genes and Genomes; AVO, acidic vesicular organelles; 2-DG, 2-deoxy-D-glucose; H2DCFDA, 2′, 7′ -dichlorodihydrofluorescein; NAC, N-acetyl-cysteine; p70S6K, p70 ribosomal protein S6 kinase; MFFs, mouse embryonic fibroblast; CQ, chloroquine; TCGA, The Cancer Genome Atlas; TLR4, Toll-like receptor 4; miRNAs, microRNAs; HIF, hypoxia-inducible factor; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma.
Yu-Ting Kang and Wen-Cheng Hsu: performed experiments, analyzed data and literature search.
Chih-Hsien Wu, I-Lun Hsin and Pei-Ru Wu: analyzed data and contributed to writing of the manuscript.
Kun-Tu Yeh and Jiunn-Liang Ko: designed the study and supervised the manuscript preparation.
All authors were involved in preparing the paper and had final approval of the submitted and published versions.
RNAi reagents were obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Center, Academia Sinica), which is supported by the National Research Program for Genomic Medicine, Ministry of Science and Technology Grant NSC-97-3112-B-001-016. Confocal microscopy, flow cytometry and use of microplate reader were performed at the Instrument Center of Chung-Shan Medical University, which is supported in part by the Ministry of Science and Technology, Ministry of Education, and Chung Shan Medical University.
The authors declare no potential conflicts of interest.
This work was supported by the Ministry of Science and Technology, R.O.C. (MOST 103-2320-B-040 -003 -MY3 and 105-2320-B-040 -014 -MY3). Flow cytometry and use of microplate reader were performed at the Instrument Center of Chung-Shan Medical University.
1. Doria A, Gatto M, Punzi L. Autophagy in human health and disease. N Engl J Med. 2013; 368:1845.
2. Goldsmith J, Levine B, Debnath J. Autophagy and cancer metabolism. Methods Enzymol. 2014; 542:25-57.
3. Kasprzak KS, Sunderman FW Jr, Salnikow K. Nickel carcinogenesis. Mutat Res. 2003; 533:67-97.
4. Kuo CY, Wong RH, Lin JY, Lai JC, Lee H. Accumulation of chromium and nickel metals in lung tumors from lung cancer patients in Taiwan. J Toxicol Environ Health A. 2006; 69:1337-1344.
5. Beveridge R, Pintos J, Parent ME, Asselin J, Siemiatycki J. Lung cancer risk associated with occupational exposure to nickel, chromium VI, and cadmium in two population-based case-control studies in Montreal. Am J Ind Med. 2010; 53:476-485.
6. Arita A, Costa M. Epigenetics in metal carcinogenesis: nickel, arsenic, chromium and cadmium. Metallomics. 2009; 1:222-228.
7. Yao Y, Costa M. Toxicogenomic effect of nickel and beyond. Arch Toxicol. 2014; 88:1645-1650.
8. Wu CH, Tang SC, Wang PH, Lee H, Ko JL. Nickel-induced epithelial-mesenchymal transition by reactive oxygen species generation and E-cadherin promoter hypermethylation. J Biol Chem. 2012; 287:25292-25302.
9. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324:1029-1033.
10. Wilson JE. Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol. 2003; 206:2049-2057.
11. Pedersen PL. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr. 2007; 39:211-222.
12. Ogawa H, Nagano H, Konno M, Eguchi H, Koseki J, Kawamoto K, Nishida N, Colvin H, Tomokuni A, Tomimaru Y, Hama N, Wada H, Marubashi S, et al. The combination of the expression of hexokinase 2 and pyruvate kinase M2 is a prognostic marker in patients with pancreatic cancer. Mol Clin Oncol. 2015; 3:563-571.
13. Palmieri D, Fitzgerald D, Shreeve SM, Hua E, Bronder JL, Weil RJ, Davis S, Stark AM, Merino MJ, Kurek R, Mehdorn HM, Davis G, Steinberg SM, et al. Analyses of resected human brain metastases of breast cancer reveal the association between up-regulation of hexokinase 2 and poor prognosis. Mol Cancer Res. 2009; 7:1438-1445.
14. Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell. 2014; 53:521-533.
15. Bolignano D, Coppolino G, Lacquaniti A, Buemi M. From kidney to cardiovascular diseases: NGAL as a biomarker beyond the confines of nephrology. Eur J Clin Invest. 2010; 40:273-276.
16. Rodvold JJ, Mahadevan NR, Zanetti M. Lipocalin 2 in cancer: when good immunity goes bad. Cancer Lett. 2012; 316:132-138.
17. Mongre RK, Sodhi SS, Sharma N, Ghosh M, Kim JH, Kim N, Park YH, Shin YG, Kim SJ, Jiao ZJ, Huynh do L, Jeong DK. Epigenetic induction of epithelial to mesenchymal transition by LCN2 mediates metastasis and tumorigenesis, which is abrogated by NF-kappaB inhibitor BRM270 in a xenograft model of lung adenocarcinoma. Int J Oncol. 2016; 48:84-98.
18. Zhang W, Yang S, Cui L, Zhang J. Neutrophil gelatinase-associated lipocalin worsens ischemia/reperfusion damage of kidney cells by autophagy. Ren Fail. 2016; 38:1136-1140.
19. Jin D, Zhang Y, Chen X. Lipocalin 2 deficiency inhibits cell proliferation, autophagy, and mitochondrial biogenesis in mouse embryonic cells. Mol Cell Biochem. 2011; 351:165-172.
20. Pierotti MA, Berrino F, Gariboldi M, Melani C, Mogavero A, Negri T, Pasanisi P, Pilotti S. Targeting metabolism for cancer treatment and prevention: metformin, an old drug with multi-faceted effects. Oncogene. 2013; 32:1475-1487.
21. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin and reduced risk of cancer in diabetic patients. BMJ. 2005; 330:1304-1305.
22. Decensi A, Puntoni M, Goodwin P, Cazzaniga M, Gennari A, Bonanni B, Gandini S. Metformin and cancer risk in diabetic patients: a systematic review and meta-analysis. Cancer Prev Res (Phila). 2010; 3:1451-1461.
23. Kasznicki J, Sliwinska A, Drzewoski J. Metformin in cancer prevention and therapy. Ann Transl Med. 2014; 2:57.
24. Anisimov VN. Metformin for cancer and aging prevention: is it a time to make the long story short? Oncotarget. 2015; 6:39398-39407. https://doi.org/10.18632/oncotarget.6347.
25. Horak E, Zygowicz ER, Tarabishy R, Mitchell JM, Sunderman FW Jr. Effects of nickel chloride and nickel carbonyl upon glucose metabolism in rats. Ann Clin Lab Sci. 1978; 8:476-482.
26. Chen H, Costa M. Effect of soluble nickel on cellular energy metabolism in A549 cells. Exp Biol Med (Maywood). 2006; 231:1474-1480.
27. Wu D, Yotnda P. Production and detection of reactive oxygen species (ROS) in cancers. J Vis Exp. 2011.
28. Candido S, Abrams SL, Steelman LS, Lertpiriyapong K, Fitzgerald TL, Martelli AM, Cocco L, Montalto G, Cervello M, Polesel J, Libra M, McCubrey JA. Roles of NGAL and MMP-9 in the tumor microenvironment and sensitivity to targeted therapy. Biochim Biophys Acta. 2016; 1863:438-448.
29. Logie L, Harthill J, Patel K, Bacon S, Hamilton DL, Macrae K, McDougall G, Wang HH, Xue L, Jiang H, Sakamoto K, Prescott AR, Rena G. Cellular responses to the metal-binding properties of metformin. Diabetes. 2012; 61:1423-1433.
30. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012; 149:274-293.
31. Meijer AJ, Codogno P. Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol. 2004; 36:2445-2462.
32. Guo H, Chen L, Cui H, Peng X, Fang J, Zuo Z, Deng J, Wang X, Wu B. Research advances on pathways of nickel-induced apoptosis. Int J Mol Sci. 2015.
33. Ballaz S, Mulshine JL. The potential contributions of chronic inflammation to lung carcinogenesis. Clin Lung Cancer. 2003; 5:46-62.
34. Kawanishi S, Inoue S, Oikawa S, Yamashita N, Toyokuni S, Kawanishi M, Nishino K. Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds. Free Radic Biol Med. 2001; 31:108-116.
35. Rothenberg ME. Innate sensing of nickel. Nat Immunol. 2010; 11:781-782.
36. Chiou YH, Wong RH, Chao MR, Chen CY, Liou SH, Lee H. Nickel accumulation in lung tissues is associated with increased risk of p53 mutation in lung cancer patients. Environ Mol Mutagen. 2014; 55:624-632.
37. Chiou YH, Liou SH, Wong RH, Chen CY, Lee H. Nickel may contribute to EGFR mutation and synergistically promotes tumor invasion in EGFR-mutated lung cancer via nickel-induced microRNA-21 expression. Toxicol Lett. 2015; 237:46-54.
38. Harty LC, Guinee DG Jr, Travis WD, Bennett WP, Jett J, Colby TV, Tazelaar H, Trastek V, Pairolero P, Liotta LA, Harris CC, Caporaso NE. p53 mutations and occupational exposures in a surgical series of lung cancers. Cancer Epidemiol Biomarkers Prev. 1996; 5:997-1003.
39. Sun H, Shamy M, Costa M. Nickel and epigenetic gene silencing. Genes (Basel). 2013; 4:583-595.
40. Costa M, Davidson TL, Chen H, Ke Q, Zhang P, Yan Y, Huang C, Kluz T. Nickel carcinogenesis: epigenetics and hypoxia signaling. Mutat Res. 2005; 592:79-88.
41. Peschiaroli A, Giacobbe A, Formosa A, Markert EK, Bongiorno-Borbone L, Levine AJ, Candi E, D'Alessandro A, Zolla L, Finazzi Agro A, Melino G. miR-143 regulates hexokinase 2 expression in cancer cells. Oncogene. 2013; 32:797-802.
42. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103:2257-2261.
43. Akao Y, Nakagawa Y, Kitade Y, Kinoshita T, Naoe T. Downregulation of microRNAs-143 and -145 in B-cell malignancies. Cancer Sci. 2007; 98:1914-1920.
44. Takagi T, Iio A, Nakagawa Y, Naoe T, Tanigawa N, Akao Y. Decreased expression of microRNA-143 and -145 in human gastric cancers. Oncology. 2009; 77:12-21.
45. Fang R, Xiao T, Fang Z, Sun Y, Li F, Gao Y, Feng Y, Li L, Wang Y, Liu X, Chen H, Liu XY, Ji H. MicroRNA-143 (miR-143) regulates cancer glycolysis via targeting hexokinase 2 gene. J Biol Chem. 2012; 287:23227-23235.
46. Li LQ, Yang Y, Chen H, Zhang L, Pan D, Xie WJ. MicroRNA-181b inhibits glycolysis in gastric cancer cells via targeting hexokinase 2 gene. Cancer Biomark. 2016; 17:75-81.
47. Jiang JX, Gao S, Pan YZ, Yu C, Sun CY. Overexpression of microRNA-125b sensitizes human hepatocellular carcinoma cells to 5-fluorouracil through inhibition of glycolysis by targeting hexokinase II. Mol Med Rep. 2014; 10:995-1002.
48. Hu JW, Sun P, Zhang DX, Xiong WJ, Mi J. Hexokinase 2 regulates G1/S checkpoint through CDK2 in cancer-associated fibroblasts. Cell Signal. 2014; 26:2210-2216.
49. Salnikow K, Davidson T, Costa M. The role of hypoxia-inducible signaling pathway in nickel carcinogenesis. Environ Health Perspect. 2002; 110:831-834.
50. Chen H, Costa M. Iron- and 2-oxoglutarate-dependent dioxygenases: an emerging group of molecular targets for nickel toxicity and carcinogenicity. Biometals. 2009; 22:191-196.
51. Patra KC, Wang Q, Bhaskar PT, Miller L, Wang Z, Wheaton W, Chandel N, Laakso M, Muller WJ, Allen EL, Jha AK, Smolen GA, Clasquin MF, et al. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell. 2013; 24:213-228.
52. Takiyama Y, Harumi T, Watanabe J, Fujita Y, Honjo J, Shimizu N, Makino Y, Haneda M. Tubular injury in a rat model of type 2 diabetes is prevented by metformin: a possible role of HIF-1alpha expression and oxygen metabolism. Diabetes. 2011; 60:981-992.
53. Wang H, Wang L, Zhang Y, Wang J, Deng Y, Lin D. Inhibition of glycolytic enzyme hexokinase II (HK2) suppresses lung tumor growth. Cancer Cell Int. 2016; 16:9.
54. Wang L, Wang J, Xiong H, Wu F, Lan T, Zhang Y, Guo X, Wang H, Saleem M, Jiang C, Lu J, Deng Y. Co-targeting hexokinase 2-mediated Warburg effect and ULK1-dependent autophagy suppresses tumor growth of PTEN- and TP53-deficiency-driven castration-resistant prostate cancer. EBioMedicine. 2016; 7:50-61.
55. Bartis D, Mise N, Mahida RY, Eickelberg O, Thickett DR. Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important? Thorax. 2014; 69:760-765.
56. Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 2003; 15:740-746.
57. Li J, Yang B, Zhou Q, Wu Y, Shang D, Guo Y, Song Z, Zheng Q, Xiong J. Autophagy promotes hepatocellular carcinoma cell invasion through activation of epithelial-mesenchymal transition. Carcinogenesis. 2013; 34:1343-1351.
58. Indelicato M, Pucci B, Schito L, Reali V, Aventaggiato M, Mazzarino MC, Stivala F, Fini M, Russo MA, Tafani M. Role of hypoxia and autophagy in MDA-MB-231 invasiveness. J Cell Physiol. 2010; 223:359-368.
59. Macintosh RL, Timpson P, Thorburn J, Anderson KI, Thorburn A, Ryan KM. Inhibition of autophagy impairs tumor cell invasion in an organotypic model. Cell Cycle. 2012; 11:2022-2029.
60. Lee HG, Kim H, Son T, Jeong Y, Kim SU, Dong SM, Park YN, Lee JD, Lee JM, Park JH. Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma. Oncotarget. 2016; 7:41798-41810. https://doi.org/10.18632/oncotarget.9723.
61. Brocato J, Costa M. Basic mechanics of DNA methylation and the unique landscape of the DNA methylome in metal-induced carcinogenesis. Crit Rev Toxicol. 2013; 43:493-514.
62. Zhan Z, Xie X, Cao H, Zhou X, Zhang XD, Fan H, Liu Z. Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination. Autophagy. 2014; 10:257-268.
63. Glaros T, Fu Y, Xing J, Li L. Molecular mechanism underlying persistent induction of LCN2 by lipopolysaccharide in kidney fibroblasts. PLoS One. 2012; 7:e34633.
64. Connor AJ, Laskin JD, Laskin DL. Ozone-induced lung injury and sterile inflammation. Role of toll-like receptor 4. Exp Mol Pathol. 2012; 92:229-235.
65. Huang H, Zhu J, Li Y, Zhang L, Gu J, Xie Q, Jin H, Che X, Li J, Huang C, Chen LC, Lyu J, Gao J, Huang C. Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells. Autophagy. 2016; 12:1687-1703.
66. Son YO, Pratheeshkumar P, Divya SP, Zhang Z, Shi X. Nuclear factor erythroid 2-related factor 2 enhances carcinogenesis by suppressing apoptosis and promoting autophagy in nickel-transformed cells. J Biol Chem. 2017; 292:8315-8330.
67. Levine B. Cell biology: autophagy and cancer. Nature. 2007; 446:745-747.
68. Aredia F, Guaman Ortiz LM, Giansanti V, Scovassi AI. Autophagy and cancer. Cells. 2012; 1:520-534.
69. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000; 19:5720-5728.


Ariana Grande has officially released her new album, Sweetener. It's available to stream below via Apple Music or Spotify.
Sweetener spans 15 tracks, including early singles in "No Tears Left to Cry", the Nicki Minaj-assisted "The Light is Coming", and one of the best songs of July, "God is a Woman". In addition to Minaj, Pharrell Williams ("Blazed") and Missy Elliott ("Borderline") turn in guest contributions. Renowned Swedish producer Max Martin provides work behind the boards, as does ILYA, who previously assisted on Grande's 2014 My Everything hit "Problem".
The new LP marks Grande's fourth overall and the follow-up to 2016's widely successful Dangerous Woman. It's also the pop singer's first full-length since the 2017 Manchester Arena attack, which claimed the lives of more than 20 of Grande's concert attendees. To help raise money for the victims' families, Grande famously put together the One Love Manchester benefit concert featuring acts like Miley Cyrus, Justin Bieber, and Coldplay.
Speaking to Jimmy Fallon, Grande said Sweetener is "about bringing light to a situation or someone's life, somebody else who brings light to your life, or sweetening the situation" — perhaps a reference to her grappling with the aftermath of the Manchester attack, or her new engagement to Saturday Night Live's Pete Davidson (he does have a song named after him after all).


Selling Books/Notebooks/School Supplies & More!
Thread: Selling Books/Notebooks/School Supplies & More!
Kneeling Chair: reduces lower back strain while studying $50. Perfect for those who get back pain while studying or sitting for long periods of time.
Send me a message with your email and I'll let you know what room I am in. Thanks!
Do you still have clip boards?
hello, I am interested in the iron, the fan and the hamper if they are still available? Or if you have photos? And i know someone who is looking for a yoga mat if that is as well.
yes they are still available. what is your email address?
It is annedorj at g mail Thanks!


text:
Orange County Register, Sept. 25, 2019
Therapy dogs Pixel and Dexter bring smiles to UCI Medical Center employees
"Pet therapy has been around for a long time, and we usually go to patients," Pat Patton, chief nursing officer at UCI Health, said. "We found through the work of (the) wellness program that we could bring it to our employees as well. "It brings smiles to people's faces, it gives them that break," Patton said. "It gives them that way to just to connect to something else besides the day-to-day patient care and helps them go on and take better care of patients here at UCI." [Subscription required, you can request an electronic copy of the article by sending an email to communications@uci.edu.]
World Economic Forum, Sept. 26, 2019
AI can help us predict the size of the next forest wildfire
"A useful analogy is to consider what makes something go viral in social media," says lead author Shane Coffield, a doctoral student in earth system science at the University of California, Irvine. "We can think about what properties of a specific tweet or post might make it blow up and become really popular—and how you might predict that at the moment it's posted or right before it's posted."
Mashable India, Sept. 25, 2019
The U.N. Just Confirmed That The Ocean Is Screwed
Just this week, Arctic sea ice dropped to its second-lowest level on record, which is over 2 million square kilometers below the average minimum ice extent during previous, cooler decades. "The rapid changes in the Arctic are some of the clearest indicators of anthropogenic climate change," Zack Labe, a climate scientist and Ph.D. candidate at the University of California, Irvine, told Mashable.
Inc., Sept. 25, 2019
This Neuroscientist Was Diagnosed as a Secret Psychopath at Age 60. Here Are 3 Signs You Might Be One Too
In 2005 58-year-old UC Irvine neuroscientist James Fallon was looking at brain scans of both serial killers and his own family for two different studies he was conducting. … The brain of one family member looked a whole lot like the brains of the criminals. "I got to the bottom of the stack, and saw this scan that was obviously pathological," he told Smithsonian Magazine. Who was this secret psychopath in his own family?
Literary Hub, Sept. 26, 2019
The Freedom of Tossing The Scarlet Letter From a High School Curriculum
In my English courses at UC Irvine, I had fallen in love with novels, stories, and writers that I could never have dreamed of back in my high school English class. … Langston Hughes's The Ways of White Folks featured breathtaking prose and stories that opened my eyes to a vast world of racial dynamics far more complicated than the bubble in which I had grown up in Orange County.

redpajama_set_name: RedPajamaCommonCrawl


PD In Focus® is an online platform that provides educators with videos about how to adopt and put into action research-based teaching practices. Depending on their roles, users can switch from a teacher view to an administrator view. Each view allows users to perform similar activities like creating playlists, watching videos, creating journals... or role-specific activities like creating accounts, managing users, and making assignments. PD In Focus® can be used collaboratively or independently.
In the first phase of this project, the platform's layout was redesigned completely. We incorporated icons and a new color palette to convey the idea that PD In Focus® is a user-friendly and energetic educational tool packed with valuable resources for educators.


Tecnopol has been present since more than 35 years in the market of distribution, compounding and trading of plastic materials.
Sales organization is supported by advanced information technologies and is carried out through an e-commerce online platform: eplast.it.
Founding year of POLIRESINE: Production of polypropylene compounds.
Founding year of Tecnopol: distribution thermoplastic resins in the northwest of Italy.
Sale of POLIRESINE and its POLIPLEN trademark to MONTELL.
of over 15 years of POLIRESINE production.
A new corporate expansion policy starts, guided by a new generation management.
The COMPOUNDING area is strengthened through the building of state of the art production facilities in Robassomero (TO), for the manufacturing of PP COMPOUND (8000 t of initial capacity).
Distribution organization expands, reaching total coverage of the Italian market with an efficient and vibrant trading network.
TECNOPOL'S TRADING also sector reinforces its market positioning, signing new and important distribution contracts.
TECNOPOL increases, almost doubling, its production capacity, reaching around 22000 t/year of PP and 4000 t/year of dry-blend PA, owing to investments in the automation of new and innovative production facilities.
As well as meeting production growth goals (24000 t/Y PP and 5000 t/Y PA), Tecnopol confirms itself as a state of the art company, veering towards e-commerce for its products through the EPLAST portal, where you can look up, order, and purchase our products online.
Tecnopol celebrates its 25 years of business.
Tecnopol celebrates its 30 years of business and get the BASF distribution for styrene in Spain.
Tecnopol gets the distribution of Ineos polyolefin products for Bulgaria and Romania.
Tecnopol strengthen the european sales network with new direct sales people in Spain and in France.


combining technology with decoration in home appliances and thus making them a significant part of our interior design scheme. Imagine your washing machine as an element of your room decoration! Or your extractor hood as an elegant livingroom centerpiece!
The new limited edition Steam Direct Washing Machine from LG and Designers Guild combines state of the art home technology with cutting-edge interior style. The collaboration has produced four eye-catching home appliances created to challenge the design and function of traditional white goods.
Elica's Star extractor hood blurs the boundaries of light and air, extraction and illumination. Star's highly decorative spherical form, an entrancing cascade of crystal, is composed of hundreds of tiny glass prisms that make it a sophisticated and fascinating light source.


But it turns out that it's a feature that can be exploited by spammers and scammers. Spammers want to get their unwanted messages into your email inbox, but as anti-spam filters have improved their chances of getting your eyeballs on their messages have reduced over the years.
This inventive spammer has used the "Add comment moderator" feature of YouTube to send me a scam message, claiming that I have "win" (sic) an Apple iPhone X.
Lucky you! Have Win Apple iPhone X Get it From : – [URL] has made you a moderator on their channel. As a moderator, you can now remove unwanted comments from videos posted on that channel. Comments you remove will be sent to the creator for their review.
How did they get their spam message injected into YouTube's standard "you've been added as a moderator" email? After all, the email really *does* come from YouTube (making it unlikely to be blocked by spam filters) and it *does* point to a YouTube channel.
It's simple. The spammer called their channel the rather ungainly "Have Win Apple iPhone X Get it From", and then included the URL they want their intended victim to click on. All in the actual name of the channel!
Meanwhile the "t.co" link will send you, via Twitter's URL shortening service, to a third-party site that definitely isn't friendly. It may be designed to steal your personal information, trick you into signing up for a bogus competition, lead to a webpage harbouring malware, or simply try to sell you something you're not interested in.
Yes, it's inventive. But it's also really rather desperate. The fact that spammers are having to use crazy tricks like this to improve their chances of having their scammy messages seen by humans warms the cockles of my heart.
Oh, and yes, YouTube has now removed the offending channel.
If you receive similar messages, report them and the channel to YouTube so the user can be banned.


Edmond Halley was already a well-known astronomer, mathematician and natural philosopher when, in 1698, he took command of His Majesty's Ship Paramore. The purpose of Halley's first two voyages was to test his geomagnetical theories; in the third voyage he investigated tidal phenomena. These voyages, which received royal approval, support from the Navy, and sponsorship from the Royal Society have been regarded as the first sea journeys undertaken for purely scientific purposes. Halley's departure on the first voyage was delayed because of his employment by Newton; and by Tsar Peter's desire to sail in the newly-commissioned Paramore. The first voyage ended prematurely with the court martial of Halley's first lieutenant. However, the second voyage was an eminently successful undertaking in which the Paramore made a wide sweep through the Atlantic reaching 52°24´S latitude. Halley made many observations which enabled him to produce the first isogonic map: this is also the earliest isoline map of any phenomenon. In his third and last voyage in the Paramore, in the summer of 1701, Halley made a detailed study of the English Channel. This voyage was also productive of highly original cartographic work including the first tidal chart. In this book the journals of Halley's three voyages are published in full from manuscripts now in the British Library. The journal of the third voyage is here published for the first time, and the earlier two are compared with versions published by Alexander Dalrymple in 1773 and 1775, from different source materials. All are extensively footnoted and preceded by a scholarly introduction by Professor Thrower on the multi-faceted aspects of Halley's important voyages. The work also contains well over one hundred documents relating to the voyages, many published for the first time. The two most original but now rare maps arising from these journeys, the Atlantic Chart and the Channel Chart (in two sheets), are reprinted in facsimile for the portfolio. Other maps, portraits, drawings, etc. illustrate this latest work from the Hakluyt Society.


Anthony's mantras are "No Space. No Equipment. No Problem." and "Anytime, Anywhere."
Anthony Lynch is a qualified fitness instructor who's been in the industry for nearly 20 years! As well as training celebrities and sports start all over the UK and Ireland, he has been a gym owner, lecturer and master coach.
Anthony is qualified in Poliquin Strength & Conditioning, S.A.Q , Agatsu Kettlebells, Indoor Cycling, Boxfit, Circuits, Animal Flow, Zuu and Ankorr training, he's also a Freemotion Master Coach. Aside from his professional career in fitness, Anthony has climbed Everest Base Camp and Kilimanjaro, competed in numerous triathlons, marathons and loves all things sports and fitness.
With all this experience behind him, Anthony is the resident fitness instructor for the first half of September Solos at BodyHoliday in Saint Lucia and will be ready to help you on your fitness journey and tailor make the best programmes for you!
Robin's background and passion is in dance and he has won numerous World Championships at domestic and international levels. Robin then went on to star in Broadway and London's West End before heading to Dancing With The Stars Australia and So You Think You Can Dance Holland.
His amazing talent took him to the BBC's Strictly Come Dancing from 2010-2013 as a professional dancer and he continues to be a regular reporter and co-presenter of It Takes Two.
Robin will be the resident host for the second half of September Solos.
Mila Lazar is a professional dancer, choreographer and fitness presenter who is a regular trainer at BodyHoliday in Saint Lucia. Mila was part of the internationally recognised pop group Alice DeeJay for some time before continuing her dance career performing for music artists. Some of these included Shaggy, En Vogue, Chaka Khan, Donna Summer, Los del Rio, Chris Brown and Kelly Rowland.
Over the past 10 years, Mila has also been developing her career as a fitness presenter, has worked with Sweaty Betty as well as developing her own HIIT workout concept.
Mila will be co-presenting the second half of September Solos at BodyHoliday in Saint Lucia with Robin Windsor.
7 nights from £2,349 based on one adult in a Standard Garden View Room on All-Inclusive.
Standard Garden View rooms are being booked up so to book, or for more information, give our friendly team of travel advisers a call on a call on 020 7751 0660 or fill in the form below.


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Archaeological Facts that Could Change the Way We See History
Recovered bits and pieces of the Antikythera Mechanism. Imgur
6. A Millennia-Old Device That Allowed People to Make Astronomical Predictions Decades in Advance
When researchers were able to take a good look at the Antikythera Mechanism and its innards, it became clear that the mysterious archaeological find had to be some kind of calculation device. However, whether it worked the way that Professor Price had reasoned was still up in the air. We now know that the device enabled users to tell how the skies would look for decades to come. That included the positions of the Sun and Moon, lunar phases, the paths of planets, and even eclipses. Cicero was one of several writers from antiquity who had mentioned the existence of such devices, but the Antikythera Mechanism is the only one ever recovered.
Unfortunately, the technology was lost in the Roman era, and the only known sample we know of ended up at the bottom of the sea, its secrets forgotten for two millennia. Current researchers have benefited greatly from the leaps and bounds that scanning technology has taken in recent years. In the first few decades after the Antikythera Mechanism's discovery, the device's advanced state of corrosion made it difficult for scholars to properly tell just what was inside the box, let alone decipher its significance. Observers could see that some of the mechanism's fragments contained what seemed to be Greek letters and words, but they were so obscured by corrosion that they were indecipherable.
Text inscribed on a fragment of the Antikythera Mechanism, revealed by modern scanning technology. Smithsonian Magazine
5. Technological Advances Finally Allowed Archaeologists to Read Text Inscribed on the Mysterious Mechanism
As one scholar stated about the Antikythera Mechanism: "Before, we could make out isolated words, but there was a lot of noise —letters that were being misread or gaps in the text … Now, we have something that you can actually read as ancient Greek. We can tell what these texts were saying to an ancient observer". That is thanks to new imaging techniques such as 3-D X-ray scans. They finally enabled researchers to legibly read about 3,400 characters that were inscribed on those parts of the device that were recovered. That is a lot, but it is only a fraction of the roughly 20,000 characters that scholars estimate had been inscribed on the archaeological find when it was whole.
The scanned texts of what was essentially a user's guide helped unravel the final mysteries of the Antikythera Mechanism. As it turned out, much of what was written amounted to an ancient version of a user's manual. For example, text inscribed on the device's back is an inventory of all of its dials, and a description of what they are for and what they signify. It is from that text that we now know that the mechanism's front had once contained a display of the planets as they moved through the zodiac.
Reconstruction of the Antikythera Mechanism. Live Science
4. A Device Commissioned by an Ancient Astronomer
The Antikythera Mechanism contained pointers with small spheres that represented the Sun, the Moon, plus Mars, Jupiter, and Saturn. All were arranged in a manner that replicated their orbit around the earth. That display did not survive the ravages of time and 2000 years' worth of salt water corrosion. Some scholars had speculated that there might have been something along those lines in the device, but those were simply educated guesses, without any hard physical proof to back them up. It was not until the device's text was finally revealed via modern scans that the guesses were transformed into concrete facts.
Other recovered texts from the archaeological find describe the risings and settings of various constellations, on different dates throughout the year. Researchers were thus able to confirm that the device's maker – or at least the person who commissioned its manufacture – was an astronomer. Researchers were also able to make out handwritten text from at least two different people. That suggests that the mechanism was not made by a single person, such as the astronomer who had recorded the sightings. Instead, it had most likely been commissioned by an astronomer, who contracted a workshop to make the device in accordance with set specifications.
Antikythera. Pinterest
3. An Archaeological Mystery Solved
The dates of celestial sightings described in the Antikythera Mechanism's text allowed scholars to identify the location of the astronomer who owned it: somewhere around latitude 35 degrees north. That is too far north for the astronomer to have been in Egypt, and too far south for him to have been in northern Greece. However, 35 degrees is a near perfect match for somebody who made observations from the Aegean island of Rhodes, just off the southwestern coast of modern Turkey. Knowledge of where the device was made (Rhodes), and where the vessel that carried it sank (off Antikythera), allows an educated guess that it had been destined for a buyer in northwestern Greece.
The mystery of the hitherto mysterious archaeological find has thus, by and large, been solved. However, there is no certainty yet about just what the device was for. As described by a scholar: "We know what it did now pretty well, but why would someone want to have something like this made? For my part, I think this is something that is very likely to have been made as an educational device, something that was not for research but for teaching people about cosmology and all sorts of time-related things about our world". Others argue that it was intended for use in astrology: to predict the movement of the stars and constellations, in an attempt to predict the future. That is still up for debate.
Ptolemaic Egypt army parade. Imgur
2. The Demise of Ancient Egypt's Language
After Alexander the Great conquered Egypt in 332 BC, it became a Hellenized kingdom ruled by the Macedonian Ptolemaic Dynasty, and Greek became the language of the rulers and the elites. Native Egyptian writing, whether colorfully picturesque hieroglyphs found on the walls of temples and monuments, or the simpler Demotic script used in everyday life, went into a steady decline. Hieroglyphs continued to be used by priests, and Demotic continued to be used by commoners. However, as century succeeded century with Egypt ruled by outsiders who used a foreign official language, native Egyptian writing waned.
The spread of Christianity centuries later eventually killed off the Ancient Egyptian religion. As the old gods' priests vanished into history, so did their knowledge of hieroglyphs. Centuries later, the arrival of Islam, Arabs, and the Arabic language, killed off the Egyptian Demotic language and script as well. Eventually, the day arrived when knowledge of Ancient Egyptian writing vanished altogether. Egypt became a country that teemed with ancient monuments, covered with colorful and intriguing texts and symbols that nobody could make head or tails of. That finally changed in the nineteenth century.
The discovery of the Rosetta Stone. Look and Learn
1. An Archaeological Discovery That Finally Gave Us a Window to Ancient Egypt
Pierre Bouchard was a French army captain who had accompanied Napoleon's expedition to Egypt. On August 21st, 1799, he was in charge of the restoration of an old fort near the town of Rosetta. When his men uncovered a block of basalt that measured 3 feet 9 inches high by 2 feet 4 inches wide, and inscribed with three different types of writing, Bouchard immediately grasped its significance. He promptly alerted a team of French scholars who had accompanied Napoleon to Egypt. The archaeological find, which came to be known as the Rosetta Stone, contained Greek, Egyptian hieroglyphics, and Egyptian Demotic scripts.
Nobody knew how to read hieroglyphs or Demotic, but scholars could read Greek. The Greek text informed archaeologists that the stone honored the second century BC King Ptolemy V. More importantly, the Greek text declared that the three scripts contained the identical message. The artifact thus held the key to solving the riddle of ancient Egyptian writing, which had been dead for over a millennium. Several scholars made initial progress in cracking the hieroglyphs, until French scholar Jean-Francois Champollion conclusively cracked the code in 1822. From then on, the language, history, and culture of ancient Egypt was opened to scholars as never before.
Adams, Mark – Meet Me in Atlantis: My Obsessive Quest to Find the Sunken City (2015)
Ancient History Encyclopedia – Troy
Ancient Origins – Questioning the Mycenaean Death Mask of Agamemnon
Antikythera Mechanism Research Project – Project Overview
Booth, Charlotte – The Boy Behind the Mask: Meeting the Real Tutankhamun (2007)
Discovering Ancient Egypt – Mystery of the Rosetta Stone
Dodson, Aidan – Amarna Sunset: Nefertiti, Tutankhamun, Ay, Horemheb, and the Egyptian Counter-Reformation (2009)
Encyclopedia Britannica – Akhenaten, King of Egypt
Encyclopedia Britannica – Sir Austen Henry Layard
Federer, Kenneth L. – Encyclopedia of Dubious Archaeology: From Atlantis to the Walum Olum (2010)
History – 6 Secrets of King Tut
History Collection – Archaeologists Find New Evidence of a Skull Cult in the World's Oldest Temple
History Extra – 8 Things You (Probably) Didn't Know About Tutankhamun
History Today, Volume 61, Issue 7, July, 2011 – The Discovery of Machu Picchu
Independent, The, September 21st, 2015 – Tutankhamun Interred in Second-hand Sarcaphagus: Researchers Discover Ornate Stone 'Coffin' Was Altered For Boy King After Egypt Turned Against Earlier Pharaoh
Live Science, June 23rd, 2016 – Ancient Greek 'Computer' Came With a User Guide
Live Science – 'Lost City' of Atlantis: Facts & Fable
National Geographic – Atlantis
National Geographic, February 17th, 2010 – King Tut Mysteries Solved: Was Disabled, Malarial, and Inbred
National Geographic, June 19th, 2017 – Cats Domesticated Themselves, Ancient DNA Shows
New Scientist, September 10th, 2015 – New Species of Extinct Human Found in Cave May Rewrite History
Proceedings of the National Academy of Sciences, January 7th, 2014 – Earliest Evidence For Commensal Processes of Cat Domestication
Smithsonian Magazine, February, 2015 – Decoding the Antikythera Mechanism, the First Computer
Smithsonian Magazine, June 8th, 2016 – The World's First Computer May Have Been Used to Tell Fortunes
Vox, May 17th, 2017 – The Antikythera Mechanism is a 2000 Year Old Computer
Wikipedia – Homo Naledi
Wikipedia – Tutankhamun


These Pyle Hydra wakeboard speakers are constructed with marine applications in mind – they're water resistant and would work great on the dock or at sea. These 300-watt speakers are equipped with an 8" poly mica woofer cone for a booming low end and 1" polymer dome tweeter for sweet highs for a frequency response of 60 Hz to 20 kHz. Butyl rubber surround and heavy duty plastic cabinet construction extends the life of your product, and gold plated plug terminals keep your sound distortion-free. Mounting bracket included.
These Pyle Hydra wakeboard speakers are constructed with marine applications in mind � they�re water resistant and would work great on the dock or at sea. These 300-watt speakers are equipped with an 8� poly mica woofer cone for a booming low end and 1� polymer dome tweeter for sweet highs for a frequency response of 60 Hz to 20 kHz. Butyl rubber surround and heavy duty plastic cabinet construction extends the life of your product, and gold plated plug terminals keep your sound distortion-free. Mounting bracket included.


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A Striking Partnership that is Breaking Down Barriers Through Football
Rapport Building and Communicating Employer or Deployer
Leicestershire Police Force, Leicester City FC and the Premier League have formed a strategic partnership that uses football to unite, educate and engage young people living in the most deprived areas of the city. Blake Richardson examines the delivery methods of Leicester City FC Community Trust and the youth engagement strategies the Police are using to reduce crime and increase community cohesion
Football can make a big difference to people's lives. It is our national sport, after all, stirring unbridled passion and human emotion in its rawest form.
Exactly how much of a transformative impact it can have on people's lives is astonishing.
The full scale of football's power to engage, unite and educate was revealed in a presentation at the 2017 UK Coaching Summit publicising the strategic partnership between Leicestershire Police Force, Leicester City FC and the Premier League.
Since joining the Premier League Kicks programme, Leicester City FC Community Trust has set up free football sessions in the most impoverished areas of the city.
The activities run in alignment with Leicestershire Police's youth crime strategies, designed to help disaffected inner-city youths make more positive life choices.
Through the parallel Community Champions initiative, which offers informal sports education workshops, qualifications and courses to those from lower socio-economic backgrounds, scores of young people have been given opportunities for employment and a foot up onto the career ladder.
They have become role models in the process, encouraging others living in deprived pockets of the community not to succumb to a life of crime.
By joining forces, the three-way community alliance has maximised the impact of using the beautiful game as an enabling tool to engage those from hard-to-reach, high-need and at risk areas of the city with a view to bettering their chances in life.
Crime prevention strategy
The diverse areas we targeted were seeing knife crime, guns and gang wars and the cohesion of the community was not there. In these types of areas, it is young people versus the older people and young people versus the police, leading to jail, drugs and death."
A graphic synopsis of the problems facing inner-city communities in Leicester, provided by PC Rob Evans, a Neighbourhood Officer (or 'beat bobby' in old school language) who has been involved with the Kicks project since its inception in 2014.
These young people are on the precipice of making the wrong choices in life. Some of them have already made the wrong choices,' he added.
"They live in a cocoon lifestyle. We are taking them beyond that and it's all having a valuable impact."
This impact can be measured in monetary terms to the tune of £100,000 a year per person.
This is the estimated outlay incurred (according to a costing exercise undertaken by PC Evans) of sending a single offender through the judicial process, culminating in a prison sentence and re-education following their release.
"The police force has reduced budgets so we need to be smarter in the way we work in terms of engaging with young people and critical communities."
Reducing the cost of crime by breaking the vicious 'prison-reoffending-prison' cycle is a major priority, but the economic benefits are secondary to the social and psychological impact the project is having – in terms of people's personal growth, well-being and career prospects and the long-term effect on community cohesion and society as a whole.
Four pillars of youth work
Cevil Bishop is the second half of the double act. Known as CJ to those he works with, he is a youth worker for LCFC Community Trust.
He hails from the same tough, austerity-plagued streets as those he now helps mentor, having grown up in the densely populated St Matthews inner-city estate, one of the poorest communities in the UK.
He says his background helps him form an initial contact.
And CJ has another thing in common with many of Leicester's young housing estate residents: a love of sport – having played professional basketball in Serbia before he began to work with young people as a behaviour mentor.
Cevil Bishop, better known as CJ
Once you've got them on board, and if you can tap into where they want to go, and what their talents are, then you can put them on that positive path and help them think more critically about enhancing their learning and their educational experience."
CJ started at the Trust in his second year at university, where he studied Youth Work and Community Development and where he learned that underpinning all work undertaken with young people is the principle that 'all practice is educative, expressive, participative and empowering'.
These four pillars of youth work are intrinsic to developing successful engagement strategies.
You have to create an environment where they want to be. Young people don't necessarily want to play football every week, but they do want to feel safe and they do want to come and talk. It's about empowerment and ownership.
"Giving them roles and peer mentor status within their satellite centres is key to growing numbers and keeping them engaged and, ultimately, in seeing them flourish. It is important in my role, therefore, to apply your softer skills."
Getting your kicks
So while CJ provides important input on life skills, organises the football and coaching sessions, and designs pathways that divert participants towards taking courses and qualifications, PC Evans and his team deliver workshops to young people on knife crime, drug-related crime, hate crime, race-related crime and look to build greater understanding around sensitive areas like stop and search laws.
A hearts and minds double whammy that is so much more than football, but with football as the alluring hook.
CJ sums it up neatly: "I have to design constructive pathways and exit routes to progress, so that the young people we target may become the positive versions of themselves that we know they are well capable of being.
"But we couldn't do this on our own. The attraction, of course, is Leicester City Football Club."
The Foxes' success helped entice 1,800 youngsters from the city through the Premier League Kicks turnstiles in one year, meaning the number of venues had to be expanded from five to 20.
Leicester winning the Premier League helped with recruitment, and the fact the first-team players have bought into the programme has had a positive influence on our young people, but it is the opportunities they are getting which is making them stay," said CJ.
The Foxes' fairy tale 2015-16 season, when they stunned the world of football by winning the Premier League title, inspired a generation of young people eager to become the next Jamie Vardy or Riyad Mahrez.
The history boys' inspirational achievement was great news for Leicestershire Police too, providing exciting opportunities that have helped them foster better relationships with those on the scheme.
"We will use our police transport to take the Community Champions to tournaments and to Premier League away games. It is a big carrot," says PC Evans (pictured in the image at the top of the page, fourth from the left). "We are now on first-name terms with a lot of them, who shake us by the hand. So this for us is a win-win project."
The partnership programme has opened doors for disaffected young adults which they could never have anticipated before joining the project.
Three peer mentors who live in the St Matthews area of the city are now employed as Premier League Kicks coaches and another has worked for the LCFC Community Trust as a coach for the programme and as an ambassador for his community.
From 2014 to the present, the Premier League Kicks programme has engaged:
2,240 young people in free football activities
157 volunteers (young leaders)
127 qualifications completed – FA Level 1, First Aid, Junior Football Leaders
14 participants employed at LCFC (Match Day Hospitality and Club Shop).
The Community Trust measured outcomes of the Kicks programme by asking those who participated to fill in a questionnaire, the results of which were:
Do you feel safer in your community – 77.5%
Improved your skills in and away from football – 82.5%
More resilient – 90%
Relationship with Leicestershire Police – 17.5% pre-Kicks / 5% post-Kicks
Awareness of consequences of anti-social behaviour, drugs, and other negative health behaviours – 85%
And the final word to two of the participants who benefited from the project:
My overall outlook on life has improved… volunteering with LCFC and the youth service has empowered me and made me more employable."
Kicks has given me the opportunity to get my life back on track. I was regularly excluded from school and the support I received from LCFC Community Trust helped me find a job and a college course."
Evidence that the programmes have inspired no shortage of champions in the community, and testimony too of the sterling work being done by Leicester Community Trust and Leicestershire Police – who are themselves the embodiment of community champions.
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