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Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo
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Jui-Chieh Chen1,*, Ming-Ju Hsieh2,3,4,*, Chih-Jung Chen5,6,7, Jen-Tsun Lin8, Yu-Sheng Lo2, Yi-Ching Chuang2, Su-Yu Chien9,10,11, Mu-Kuan Chen12
1Department of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan
2Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
3School of Optometry, Chung Shan Medical University, Taichung, Taiwan
4Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan
5Department of Surgical Pathology, Changhua Christian Hospital, Changhua, Taiwan
6Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan
7School of Medicine, Chung Shan Medical University, Taichung, Taiwan
8Hematology & Oncology, Changhua Christian Hospital, Changhua, Taiwan
9Department of Pharmacy, Changhua Christian Hospital, Changhua, Taiwan
10College of Health Sciences, Chang Jung Christian University, Tainan, Taiwan
11Center for General Education, Mingdao University, Changhua, Taiwan
12Department of Otorhinolaryngology-Head and Neck Surgery, Changhua Christian Hospital, Changhua, Taiwan
*These authors have contributed equally to this work
Su-Yu Chien, email: email@example.com
Mu-Kuan Chen, email: firstname.lastname@example.org
Keywords: Polyphyllin G, nasopharyngeal carcinoma, apoptosis, autophagy, MAPK
Received: March 22, 2016 Accepted: August 24, 2016 Published: September 02, 2016
Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch, has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines. Previous studies found that Polyphyllin G induced apoptotic cell death in human hepatoblastoma cancer and lung cancer cells. However, the underlying mechanisms of autophagy in human nasopharyngeal carcinoma (NPC) remain unclear. In this study, Polyphyllin G can potently induced apoptosis dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression in different human NPC cell lines (HONE-1 and NPC-039). The amount of both LC3-II and Beclin-1 was intriguingly increased suggest that autophagy was induced in Polyphyllin G-treated NPC cells. To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were combined treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125). These results demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy. Finally, an administration of Polyphyllin G effectively suppressed the tumor growth in the NPC carcinoma xenograft model in vivo. In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma.
Nasopharyngeal carcinoma (NPC) is a cancer that arises from the epithelium of nasopharynx. Although such cancer occurs rarely in Europe and America, its incidence is much higher in Africa and Southeast Asia . Advanced staging is the most important prognostic factor worldwide in NPC, decreasing 5-year overall survival rate from 100%-86.1% for Stage I-II disease to 50.7% for Stage IV disease . Its unique epidemiology, pathogenesis and relation to the Epstein–Barr virus make it different to other head and neck cancers . Although the cancer is known for its good response to radiation therapy, chemotherapy still has its role in current protocol.
Apoptosis and autophagy are discrete cellular processes that are mediated by distinct groups of regulatory and executioner molecules [4, 5]. Apoptosis is type I form of programmed cell death that is executed by activated caspases, which are speciﬁc enzymes that participate in signaling cascades that culminate in the rapid removal of organelles and other cellular structures [6, 7]. Autophagy is a major intracellular degradation mechanism that operates under stress conditions to promote survival during starvation or to cause programmed type II cell death under specific conditions (e.g., apoptosis inhibition) [8–10]. Autophagy has been characterized as both a unique cell-death pathway and an adaptation to stress that promotes cell survival. . Recently, extensive attention has been paid to the role of autophagy in cancer development and therapy [12–14]. The relationship between apoptosis and autophagy is complex, and varies according to cell types and stressors. It remains uncertain whether autophagy represents a mechanism for preventing apoptosis or for enacting non-apoptotic programmed cell death. Certain agents kill cancer cells through non-apoptotic pathways, and may circumvent chemo-resistance. Such agents may be promising for treating resistant cancers . For this reason, tumor cells with apoptosis defects undergo autophagy, so inhibiting autophagy causes tumor cells to die through alternative mechanisms . It has also been suggested that autophagy may provide a useful method to prevent cancer development, limit tumor progression, and enhance the efficacy of cancer treatments .
Many vegetables, fruits, and grains offer substantial protection against various cancers [18–20]. There is increasing focus on providing a scientific basis for using these agents as a preventive strategy for people with a high risk of cancer. Therefore, inducing the death of cancer cells using chemotherapeutic agents may help to achieve a successful chemotherapy in patients. Rhizoma Paridis mainly contains steroidal saponins, especially diosgenyl saponins and pennogenyl saponins . Several steroidal saponins possessing anticancer properties against a variety of cancer cells have been isolated and identified from Rhizoma Paridis . Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch [23–25]. Polyphyllin G has been demonstrated to have strong anticancer activities via suppression of proliferation, cell cycle arrest, and modulation of drug resistance in a wide variety of human cancer cell lines [24, 26–29]. Although Polyphyllin G exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms of autophagy in human NPC remain unclear. The aim of this study was to characterize the effects of Polyphyllin G on human NPC cells, and elucidate the underlying molecular mechanism responsible for autophagy and apoptosis in Polyphyllin G-induced cytotoxicity.
Cytotoxic effects of Polyphyllin G on human NPC cell lines
The chemical structure of Polyphyllin G is shown in Figure 1A. To assess the effects of Polyphyllin G on cell viability, HONE-1 and NPC-039 cells were cultured in the presence of increasing concentrations of Polyphyllin G for 24 h. As shown in Figure 1B-1C, Polyphyllin G significantly inhibited cell viability in a dose-dependent manner. Polyphyllin G (4 μM) also substantially decreased the cell viability of HONE-1 and NPC-039 cells in a time-dependent fashion, compared with untreated cells (Figure 1D). To further investigate the anti–cell-growth activity of Polyphyllin G, a clonogenic assay was performed to determine the long-term effect of Polyphyllin G treatment on NPC cancer cells. Polyphyllin G (25 μM) significantly inhibited the colony-formation ability of HONE-1 and NPC-039 cells (Figure 1E). These results indicated that Polyphyllin G can potently inhibit cell viability of different human NPC cell lines.
Figure 1: Polyphyllin G reduces cell viability in the dose- and time-dependent manners. A. Chemical structure of Polyphyllin G. B. HONE-1 and C. NPC-039 cells were treated with indicated concentrations of Polyphyllin G for 24 h, after which the cell viability was measured using MTT assay. D. Cells cultured in the presence of Polyphyllin G (4 μM) for 6, 12 and 24 h, respectively. Cell viability was measured by MTT assay. Results are shown as mean ± SD from 3 determinations per condition repeated 3 times. *P < 0.05, compared with the control (0 μM or 0 h). E. Equal numbers of cells from the Polyphyllin G-treated HONE-1 and NPC-039 cell pools were plated and stained as described in the text. The number of colonies was counted under a dissecting microscope. The data show the relative colony number, and the number of cell lines without Polyphyllin G treatment was set at 100%. Results are shown as mean ± SE. *p < 0.05, compared with the HONE-1 (0 μM). #p < 0.05, compared with the NPC-039 (0 μM).
Polyphyllin G-induced cell cycle arrest and cell apoptosis in human NPC cell lines
To elucidate whether Polyphyllin G inhibits cell growth through the induction of apoptosis, we investigated the effects of Polyphyllin G on apoptosis in NPC cell lines. As shown in Figure 2A-2B, apoptotic cells with condensed and fragmented nuclei were gradually increased in a dose-dependent manner. We next analyzed cell cycle of Polyphyllin G-treated HONE-1 and NPC-039 cells. We observed a dose-dependent increase of the sub-G1 population, as assessed by flow cytometry (Figure 2C-2D). Furthermore, Annexin V/PI double staining and caspase-3/7 staining were also performed, and the results showed in a dose-dependent increase of both early and late apoptotic cells (Figure 2E-2F). To visualize the apoptotic features, cells were stained with JC-1. In the fluorescent images, a dose-dependent increase of green signal was detected in the cells treated with Polyphyllin G. The mitochondrial membrane potential was reduced in Polyphyllin G-treated NPC cell lines (Figure 3A-3B). To further elucidate the mechanisms of Polyphyllin G-induced apoptosis in NPC cells, we evaluated the involvement of apoptosis-related proteins in the apoptotic process by Western blot analysis. After the treatment with Polyphyllin G for 24 h, cleavage of caspase-8, caspase-3, caspase-9, and PARP significantly increased in a dose-dependent manner (Figure 3C-3D). Furthermore, Polyphyllin G also caused a dramatic dose-dependent decrease in the protein level of Bcl-2 and Bcl-xL, while Bax protein level was significantly increased (Figure 3E-3F). Collectively, these data demonstrated Polyphyllin G-induced apoptosis was dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression. To clarify the relevance of Polyphyllin G-induced cell death, z-VAD-FMK (a broad-spectrum caspase inhibitor) was used in the following experiments. Polyphyllin G combined with Z-VAD-FMK substantially increase the cell viability and decrease apoptosis cells of HONE-1 and NPC-039 cells (Figure 3G-3H).
Figure 2: Polyphyllin G induces apoptosis in HONE-1 and NPC-039 cells. A. Cells were treated with different concentration of Polyphyllin G (1-4 μM) for 24 h and then stained with DAPI. Fragmented or condensed nuclei could be observed under a fluorescence microscope as indicated by the arrows. B. Results are shown as mean ± SE from 3 determinations per condition repeated 3 times. *P < 0.05, compared with the control (0 μM). C. Cells were stained with propidium iodide (PI), and analyzed for DNA content by Muse Cell Analyzer flow cytometry. The percentages of apoptotic cells were evaluated by sub-G1 DNA content (hypodiploid DNA). D. The quantified data of cell cycle distribution. E. For quantitative analysis of apoptosis, cells were dual-labeled with PI and Annexin V fluorescence and analyzed by Muse Cell Analyzer flow cytometry. F. For quantitative analysis of apoptosis, cells were staining with Muse Caspase-3/7 assay kit and analyzed by Muse Cell Analyzer flow cytometry. Data are representative of at least 3 independent experiments. Results are shown as mean ± SE. *p < 0.05, compared with the HONE-1 (0 μM). #p < 0.05, compared with the NPC-039 (0 μM).
Figure 3: Effects of Polyphyllin G on apoptotic and anti-apoptotic protein expression in HONE-1 and NPC-039 cells. A. Cells were treated with an indicated concentration of Polyphyllin G for 24 h and subjected to staining for JC-1. Fluorescent signals were observed using a fluorescence microscope. B. Cells were treated with increasing dose of Polyphyllin G for 24 h. The mitochondrial membrane potential was investigated by Muse Cell Analyzer flow cytometry and the data was processed by the Muse®Cell Analyzer Assays. C. A representative Western blot for expression of cleaved caspase-8, -3, -9 and PARP cleavage in cells after stimulation with Polyphyllin G at different concentration. D. The values under each lane indicate relative density of the band normalized to β-actin, respectively. E. Cells were treated with indicated concentration of Polyphyllin G for 24 h and subjected to Western blot with antibodies against Bcl-2, Bcl-xL-2, and Bax. F. Band intensity was quantified by densitometry analysis. β-actin was used as an internal control to normalize the amount of proteins applied in each lane. Values represent the mean ± SD of 3 independent experiments. *P < 0.05, compared with the control (0 μM). G. HONE-1 and NPC-039 cells (5 × 104 cells/well of 24-well plate) were treated with Polyphyllin G (2 μM) in the presence or absence of apoptosis inhibitor z-VAD-FMK (20 μM) for 24 h, as analyzed by MTT assay and H. dual-labeled with PI and Annexin V fluorescence and analyzed by flow cytometry. Results are shown as mean ± SE. *P<0.05, compared with the control (0 μM). #P<0.05, compared with the Polyphyllin G treatment (2 μM).
Polyphyllin G induced autophagy via inhibition of mTOR phosphorylation in NPC cells
Although a number of commonly used anticancer drugs have shown to induce apoptosis in cancer cells, autophagy is also frequently observed in response to these treatments [30, 31]. We next investigated whether Polyphyllin G induces autophagy in NPC cells. To further confirm the involvement of autophagy in Polyphyllin G treatment, we analyzed the expression of the autophagic markers LC3I/II, Beclin-1, and the cargo receptor p62 by Western blot. As shown in (Figure 4A-4D), the amount of both LC3-II and Beclin-1 was intriguingly increased in both tested cells. Additionally, Polyphyllin G significantly decreased the level of p62. These changes in the expression levels of autophagy-related proteins indicated enhanced level of autophagy in Polyphyllin G-treated NPC cells. After 24 h of treatment with Polyphyllin G, a substantial change in LC3 puncta formation was observed, indicating the formation of autophagosomes in Polyphyllin G-treated HONE-1 and NPC-039 cells (Figure 4E). To confirm autophagic induced by Polyphyllin G, MDC staining was performed and viewed under a fluorescence microscope. MDC is a specific marker for autophagic vacuoles. Similar to Western blots, MDC-labeled autophagic vacuoles appeared as distinct dot gradually increased in a dose-dependent manner (Figure 4F). The mTOR pathway functions as an autophagy regulator under starvation or other cellular stress conditions . We detected the expression of PI3K, activated mTOR (ser2448), total mTOR, Raptor, Rictor and GβL in HONE-1 and NPC-039 cells treated with different concentrations of Polyphyllin G. As shown in Figure 4G-4H, Western blotting assays revealed that Polyphyllin G significantly decreased the expression of PI3K, activated mTOR (ser2448), total mTOR, Raptor, Rictor and GβL in a dose-dependent manner. Acidic vesicular organelle (AVO) formation (autophagosomes and autolysosomes) is a characteristic feature of autophagy  To detect the development of AVOs, untreated and treated HONE-1 and NPC-039 cells were stained with acridine orange. Polyphyllin G induced AVO formation in NPC cells (Figure 4I-4J). To clarify the relevance of Polyphyllin G-induced cell death, bafilomycin A1 (Baf A1) and wortmannin (autophagy inhibitor) was used in the following experiments. Polyphyllin G combined with Baf A1 and wortmannin substantially increase the cell viability and no effect apoptosis cells of only Polyphyllin G-treatment HONE-1 and NPC-039 cells (Figure 4K-4L).
Figure 4: Polyphyllin G induces autophagy in HONE-1 and NPC-039 cells. A. Cells were treated with indicated concentration of Polyphyllin G for 24 h and subjected to Western blot with antibodies against LC3-I/LC3-II, beclin-1, and p62. B. The intensity of bands was quantified by densitometric analysis. C. Cells were incubated with Polyphyllin G (1 μM) for the indicated time intervals and the expression levels of LC3-I/LC3-II, beclin-1, and p62 were examined by Western blot. D. The intensity of bands was quantified by densitometry analysis. E. Cells seeded onto glass coverslips with treatment with Polyphyllin G for 24 h, followed by immunostaining and an observation of LC-3 (green fluorescence) under fluorescence microscopy. Original magnifications: 200×. F Cells were treated for 24 h in the presence of Polyphyllin G (1-4 μM), and MDC was added to the medium during the last hour of culture. The quantification of MDC-stained structures was performed using the analyze particles tool of the Image J software on an average of 100 cells per treatment. The data show the mean ± SD of at least 3 independent experiments. *P < 0.05, compared with the control (0 μM). G. Cells were treated with indicated concentration of Polyphyllin G for 24 h and subjected to Western blot with antibodies against PI3K, mTOR (ser2448), mTOR, Raptor, Rictor and GβL. H. The intensity of bands was quantified by densitometric analysis. I. Cells treatment with Polyphyllin G for 24 h was stained with acridine orange for AVOs formation. Data shown are representative fluorescence microscopic photographs. Original magnifications: 200×. J. The quantification of AVOs, acridine orange-stained cells were harvested and then analyzed by flow cytometry. K. HONE-1 and NPC-039 cells (5 × 104 cells/well of 24-well plate) were treated with Polyphyllin G (2 μM) in the presence or absence of autophagy inhibitor Baf A1 (1 nM) or wortmannin (50 nM) for 24 h, as analyzed by MTT assay and L. dual-labeled with PI and Annexin V fluorescence and analyzed by flow cytometry. Results are shown as mean ± SE. *P<0.05, compared with the control (0 μM). #P<0.05, compared with the Polyphyllin G treatment (2 μM).
AKT/ERK/JNK/p38 MAPK signaling pathways are involved in Polyphyllin G-induced apoptosis and autophagy
Previously studies have reported that AKT and MAPKs signaling pathways are implicated in apoptosis and autophagy [33–36]. To further investigate the mechanism by which Polyphyllin G induces apoptosis and autophagy, we directly measured the phosphorylation of AKT and MAPKs in response to Polyphyllin G. The results revealed that stimulation of cells to Polyphyllin G induced an increase in phosphorylation of JNK1/2 in a dose-dependent manner (Figure 5A-5B). Moreover, treatment of NPC cells with Polyphyllin G (4 μM) also resulted in a time-dependent increase in phosphorylation of JNK (Figure 5C-5D). To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK1/2 inhibitor (SP600125) for 2 h followed by treatment with or without Polyphyllin G for 24h. As shown in Figure 6A, cell viability of NPC cells that pretreated with SP600125 and then treated with Polyphyllin G was increased cell viability when compared with Polyphyllin G treatment only. However, the combination of LY294002, U0126 and SB203580 with Polyphyllin G dramatically decreased cell viability in NPC cells. In addition, pre-treatment with U0126 was increased Polyphyllin G-induced apoptosis. Pre-treatment with SP600125 was decreased Polyphyllin G-induced apoptosis. However, LY294002 or SB203580 does not affect apoptosis by Polyphyllin G. Suggesting the activation of ERK1/2 and JNK1/2 was involved in cell apoptosis (Figure 6B). To address the direct role of signaling in autophagy induction, the effects of pharmacological inhibitors on the proteins levels of LC3 and p62 were examined. Our date showed that Polyphyllin G-induced autophagy was dramatically decreased in Polyphyllin G co-treatment with LY294002 or SP600125 group compared with Polyphyllin G treatment alone (Figure 6C-6D). In addition, pre-treatment with SB203580 was increased Polyphyllin G-induced autophagy suggesting the activation of AKT, p38 MAPK or JNK1/2 was involved in cell autophagy. Taken together, these results demonstrated that Polyphyllin G induced apoptosis in NPC cells through ERK1/2 reduction and activation of JNK1/2, while AKT, p38 MAPK and JNK1/2 were responsible for Polyphyllin G-induced autophagy.
Figure 5: Polyphyllin G induces the phosphorylation of AKT, ERK1/2, JNK1/2, and p38 MAPK in HONE-1 and NPC-039 cells. A. Cells were treated with different concentrations of Polyphyllin G (0-4 μM) for 24 h. The p-AKT, p-ERK1/2, p-JNK1/2, and p-p38 were analyzed by Western blot with their respective antibodies. The total protein of AKT, ERK1/2, JNK1/2, and p38 is also shown. B. Quantitative results of p-AKT, p-ERK1/2, p-JNK1/2, and p-p38 protein levels, which were adjusted with their total protein levels and expressed as multiples of induction beyond each respective control. C. Cells were incubated with Polyphyllin G (4 μM) for indicated time intervals and the phosphorylation status and total levels of proteins were measured by Western blot. D. Quantitative analyses of the phosphorylation status and total levels of proteins. Values represent the mean ± SD of at least 3 independent experiments. *P < 0.05, compared with the control (0 μM or 0 h).
Figure 6: Effects of the AKT, ERK1/2, JNK1/2, and p38 MAPK on Polyphyllin G-induced apoptosis and autophagy in HONE-1 and NPC-039 cells. Cells were pretreated with LY294002 (AKT inhibitor, 20 μM), U0126 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 MAPK inhibitor, 20 μM), or SP600125 (JNK1/2 inhibitor, 20 μM) for 1 h followed by treatment with or without Polyphyllin G for 24 h. A. Cell viability was measured by MTT assay. B. The percentages of apoptotic cells were evaluated by flow cytometry. C. The levels of expression of LC3-I/LC3-II and p62 were examined by Western blot. D. Band intensity was quantified by densitometry analysis. β-actin was used as an internal control to normalize the amount of proteins applied in each lane. Values represent the mean ± SD of 3 independent experiments. *P<0.05 vs. control (cells treated only with DMSO), and #P<0.05 vs. Polyphyllin G.
Significant antiproliferative effects of Polyphyllin G in an NPC orthotopic graft model
To test the effect of Polyphyllin G on tumor growth, the in vivo antitumor effect of Polyphyllin G was evaluated. Tumor volumes were determined by caliper measurements for every 6 days. The control group of animals transplanting NPC-039 cancer cells showed a progressive increase in their tumor volumes. In Polyphyllin G-treated mice receiving 2 and 4 mg/kg, the mean tumor volume (Figure 7A) and tumor weight (Figure 7C) were significantly inhibited compared to vehicle-treated. As illustrated in Figure 7B, no significant difference in body weight was detected among these groups. In IHC analysis for cell proliferation, Ki67-positive tumor cells were significantly reduced after treatment with Polyphyllin G compared to control mice (Figure 7D-7E). These results showed that animals treated with Polyphyllin G show anti-tumor growth as compared with control animals.
Figure 7: Polyphyllin G suppressed tumor growth NPC-039 cells in vivo. A. NPC-039 cells were injected into the right flank of 6-week-old male nude mice. After injection of NPC-039 cells, nude mice were treated with either vehicle or Polyphyllin G (2 or 4 mg/kg) for 24 days. The growth of the xenograft tumors was referred to the measurement of the long and short dimensions of the tumors, and the calculation of the tumor size were described in the ‘‘Materials and methods’’ section. B. Body weight and C. tumor weight changes of the mice during 24 days of treatment. *P < 0.05, compared to the vehicle. D. Tumor tissues were examined by H&E staining, and immunohistochemical staining with an anti-Ki67 antibody. Original magnifications: 200×. E. The mean value of the Ki67 expression percentage is indicated by a red bar. *p < 0.05, compared to the vehicle groups.
Polyphyllin G (also call Polyphyllin VII) exhibited anti-proliferative activity against the hepatocellular carcinoma HepG2 cell line [26, 37], MCF-7/ADR cell line  and lung cancer cells lines (A549 and NCI-H1299) . The present study first investigated the significant in vitro and in vivo inhibitory effects of Polyphyllin G on human nasopharyngeal cancer cells. Our data demonstrated that Polyphyllin G inhibited the growth of HONE-1 and NPC-039 cells at low IC50 values in a dose-dependent manner (Table 1). These results suggest the potent anti-cancer effects of Polyphyllin G. Various natural chemicals have been reported to exhibit anticancer activity by triggering both autophagy and apoptosis [38–41], implicated the close relationship between autophagy and apoptosis. Polyphyllin G has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines [24, 26–28]. However, the underlying mechanisms of Polyphyllin G-induced apoptosis and autophagy in human nasopharyngeal cancer cells is remain unclear.
Table 1: IC50 of Polyphyllin G in HONE-1 and NPC-039 cell lines for 24 h
IC50 (24 h)
Polyphyllin G (μM)
Previous studies show that Polyphyllin G arrested A549 and NCI-H1299 cells in the G2/M phase ; Li et al point that Polyphyllin G could induce cell apoptosis, together with cell cycle arrest in G1 phase, and trigger apoptosis in a caspase-3-dependent manner in human colorectal cancer cells (HT-29 and SW-620) . In this study, the ﬂow cytometric analysis of PI-labeled cells shows that treating NPC cell lines with Polyphyllin G (4 μM) induced signiﬁcant accumulation of cells in the sub-G1 phase (Figure 2B). The sub-G1 cell population increased to 16.9% and 14.8% in HONE-1 and NPC-039 cells treated with 4 μM Polyphyllin G for 24 h, respectively. Moreover, Annexin V/PI double staining was also performed, and the results showed in a dose-dependent increase of both early and late apoptotic cells. Previous study show that Polyphyllin G increased TNFR1, TRAIL R1/DR4, TRAIL R2/DR5, and FADD expression, and activation of PARP, caspase-8, and 3 in MCF-7/ADR cells . Zhang et al point that Polyphyllin G upregulated cleaved caspase-3, cleaved caspase-9, and Bax expression in Hela cells . Polyphyllin G treatment reduced the total Bcl-2 levels in HepG2 cells . Our data demonstrated Polyphyllin G-induced apoptosis was dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression (Figure 3). These results demonstrated that apoptosis developed in NPC-039 and HONE-1 cells after they were treated with Polyphyllin G for 24 h, and that the extent of apoptosis were related to the dose of Polyphyllin G administrated.
Autophagy is a major intracellular degradation mechanism that operates under stress conditions to promote survival during starvation or to cause programmed type II cell death under specific conditions [8–10]. Autophagy is initiated when large sections of cytoplasm are engulfed by a crescent-shaped phagophore that elongates to form an autophagosome, which subsequently fuses with a lysosome, resulting in the degradation of cellular content by lysosomal hydrolases [16, 17]. Previous study point that Polyphyllin G induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy . Polyphyllin G inhibited cell growth by down-regulating MEK1/2, ERK1/2 phosphorylation, and suppression of AKT pathway . Chen et al show that Polyphyllin G was associated with apoptosis induction and proliferation inhibition through regulation of MAPK pathway and inhibition of PI3K/Akt pathway . The present study demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy.
The present study demonstrated that Polyphyllin G suppressed the human nasopharyngeal cancer cells tumor formation in the xenograft model in vivo (Figure 7). The mean tumor volume in Polyphyllin G treated mice was smaller compared to control, however, is somewhat at odds with the in vitro data (Figure 1). Previous study show that Polyphyllin G inhibited the growth of lung cancer tumor xenografts at the dose of 1, 2, and 3 mg/kg . Present study results are consistent with the previous study and further illustrated that Polyphyllin G may also be able to apply on different cancers. The assumption credibility need more research to further prove.
In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. We believe that Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma.
MATERIALS AND METHODS
Polyphyllin G (purity > 98%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and was dissolved in dimethyl sulfoxide (DMSO) and diluted in cell culture medium to the final concentration before use. The final concentration of DMSO for all treatments was consistently less than 0.1%. All cell culture-related reagents were purchased from Invitrogen (Carlsbad, CA). DAPI dye, propidium iodide (PI), RNase A, protease inhibitor cocktail, phosphatase inhibitor cocktail, and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO). Antibody against cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), Bcl-2, Bcl-xL, Bax, LC3B, Beclin1, p62, p-AKT, AKT, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, p38, PI3K, mTOR (ser2448), mTOR, Raptor, Rictor, GβL and β-actin were purchased from Cell Signaling Technology (Danvers, MA). Specific inhibitors for AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125), z-VAD-FMK, Wortmannin and Bafilomycin A1 (Baf A1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
The human nasopharyngeal carcinoma (NPC) cell lines HONE-1 and NPC-039 cells were a gift from Dr. Jen-Tsun Lin, Hematology & Oncology, Changhua Christian Hospital (Supplemental data 1). All cells were cultured in RPMI 1640Medium (Gibco BRL, Grand Island, NY) supplemented with10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids,1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/l sodium bicarbonate, and 1 mM sodium pyruvate (Sigma, St. Louis, MO) and maintained at 37°C in a humidified atmosphere of 5% CO2.
MTT assay was used to evaluate the effect of Polyphyllin G on cell viability. Briefly, cells were seeded into 96 well plates at a density of 5000 cells/well containing 100 μl of culture medium. After overnight incubation, the cells were treated with indicated Polyphyllin G concentration. At every indicated time interval after Polyphyllin G treatment, 10 μl MTT solution was added to each well (at a final concentration of 5 mg/ml) and incubated for further 4 h. The supernatant was then discarded, and 200 μl of DMSO (dimethyl sulfoxide) was added to each well to dissolve the formazan. Optical density (OD) was evaluated by measuring the absorbance, with a test wavelength of 490 nm and a reference wavelength of 630 nm.
HONE-1 and NPC-039 cell line was seeded at a concentration of 0.05x104 cells per well in 6-well cell culture plates in appropriate media. After 24 h, media was replaced with fresh media containing either Polyphyllin G at 1, 2 and 4 μM. Medium with compound being changes every 3 days. After 2 weeks, colonies were stained with 0.3 % crystal violet solution.
Detection of apoptosis by DAPI staining
After being subjected to indicate treatment, cells were collected and fixed with 4% paraformaldehyde for 20 min. Then, cells painted on the slides and stained with DAPI dye (50 μg/ml) for 10 min. After washing with phosphate-buffered saline (PBS), the morphological changes related to apoptosis were assessed by fluorescence microscopy (Lecia, Bensheim, Germany). Percentage of apoptotic cells was scored on at least 500 cells.
In this experiment, cells were plated in six-well dishes. Following indicated drug treatments, cells fixed in 70% ethanol at −20°C for 16 h. After washing with PBS, cells were incubated for 30 min in the dark at room temperature with PI buffer (4 mg/ml PI, 1% Triton X-100, 0.5 mg/ml RNase A in PBS) and then filtered through a 40-mm nylon filter (Falcon, USA). The cell cycle distribution was measured for 5,000 events by a FACSCalibur flow cytometer (BD Biosciences) and data were analysed using CellQuest software (BD Biosciences).
Annexin V/PI double staining
We also carried out experiments using the annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Millipore) to detect the early apoptotic cells. Briefly, cells were resuspended in 100 μl 1 x binding buffer containing 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. After the addition of FITC Annexin V (2 μg/ml) and PI (2.5 μg/ml), the cell suspension was gently mixed and then incubated for 15 min at room temperature in the dark. Afterward, 400 μl of 1 x binding buffer was added to each tube followed by flow cytometry analysis within 1 h. The FITC and PI signals were measured with a flow cytometer using the CellQuest software, and 10,000 events were acquired. Quadrants were positioned on Annexin V/PI plots to distinguish living cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic/secondary necrotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+).
The levels of total apoptosis cells were detected by Muse Caspase-3/7 Assay Kit. 1×105 cells treated for 24 h with different concentrations of Polyphyllin G. After PBS washing, prepare cell samples for incubation with Muse™ Caspase-3/7 working solution at 37°C for 30 mim. Add 150 μL of Muse™ MitoPotential 7-AAD and mix thoroughly and run on Muse™ Cell Analyzer and analysis data by the Muse®Cell Analyzer Assays (Millipore).
Mitochondrial membrane potential (Δψm) measurement
The loss of Δψm was detected by JC-1 staining or Muse Mitopotential Assay Kit. For JC-1, after being subjected to indicate treatment, cells painted on the slides and stained with JC-1 staining for 10 min, 37°C. After washing with PBS, the morphological changes related to apoptosis were assessed by fluorescence microscopy (Lecia, Bensheim, Germany). For Muse Mitopotential Assay Kit, 1×105 cells treated for 24 h with different concentrations of Polyphyllin G. After PBS washing, prepare cell samples for incubation with Muse™ MitoPotential working solution at 37°C for 20 mim. Add 5 μL of Muse™ MitoPotential 7-AAD and Incubate at room temperature for 5 min. Mix thoroughly and run on Muse™ Cell Analyzer and analysis data by the Muse®Cell Analyzer Assays (Millipore).
In situ immunofluorescence assay
Cells were seeded into 6-well dish at a density of 4 × 105 cells per dish. After Polyphyllin G incubation, cells were fixed with 4% paraformaldehyde for 20 min and then incubated with 0.5% Triton X-100 for 10 min. PBS washing was conducted between each reaction to remove any residual solvent. Afterwards, fixed cells were incubated with 4% BSA at room temperature for 2 h and then with the appropriate primary antibodies at 4°C for overnight. After overnight incubation, cells were washed and then incubated with Alexa Fluor 488-conjugated affinipure goat anti-rabbit IgG secondary antibody (Jackson Immuno Research, West Grove, PA, USA) with light protection for 1 h. At the end of incubation, cells were observed under fluorescence microscopy equipped with filters for UV and Blue 488 nm.
Detection of acidic vesicular organelle (AVO) formation
Polyphyllin G-treated cells were washed with PBS, followed by staining with 1 μg/ml acridine orange for 30 min, 37°C. Afterwards, cells were washed with PBS and then observed under fluorescence microscopy equipped with filters for 488 nm. For quantification of AVOs, acridine orange-stained cells were harvested, washed twice with PBS, resuspended in PBS containing 5% FBS and then analyzed by flow cytometry.
Western blot analysis
Cells were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail. Protein concentration was determined by the BCA assay (Pierce). The equal amount of total protein was resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA). Membranes were blocked with 5% non-fat milk in PBS for 1 h and then incubated at 37°C for 1 h or at 4°C overnight with the indicated primary antibodies. Membranes were washed with PBS/0.1% Tween 20 (PBST) and incubated for 1 h at room temperature with the appropriate secondary antibodies conjugated to horseradish peroxidase. Membranes were then washed and bound antibodies were visualized by ECL detection system and relative photographic density was quantitated by a geldocumentation and analysis (AlphaImager 2000, Alpha InnotechCorporation, San Lean 189 dro, CA).
In vivo anti-tumor growth effects on xenograft transplantation
As previously described . For experimental xenograft growth inhibition study, 5-6 week male C57BL/6 mice (18–22 g) (National Taiwan University Animal Center, Taiwan) were used. NPC-039 cells (2×106 per mouse) were resuspended in 200 μl of sterile PBS and injected s.c. into the right flank of the mouse. Mice were randomized into two groups (5 mice/group). All animals were housed with a regular 12-h light/12-h dark cycle and water ad libitum access to standard rodent chow diet (LaboratoryRodent Diet 5001, LabDiet, St. Louis, MO), and kept in a pathogen-free environment at the Laboratory Animal Unit (temperature 22°C, humidity 30~70%, 5 mice/cage). Seven days after tumor cell injection, the mice were orally fed Polyphyllin G (2 mg/kg and 4 mg/kg) or vehicle three times per week. The control group received an equal volume of 0.5% carboxymethyl cellulose vehicle. Tumor volumes were determined from caliper measurements obtained every six days. At the end of the experiment, mice were animals were sacrificed and primary tumors were removed for further analysis. The primary tumors were separated from the surrounding muscles and dermis, and then weighed. The tumor volume was calculated by the following formula: 0.5 x length x width2. Mean weight of mice at initiation of study and termination of study did not differ significantly between the groups. All of the procedures involving animals were conducted in accordance with the institutional animal welfare guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Chung Shan Medical University.
Tumor immunohistochemistry (IHC)
As previously described . Paraffin embedded squamous cell carcinoma and paired non-cancerous tissue sections (4-μm) on poly-L-lysine-coated slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase activity was blocked with 3% H2O2. The antigen was retrieved by heating at 100 °C for 20 min in 10 mM citrate buffer (pH 6.0). After antigen retrieval, slides were washed with PBS and incubated with anti-Ki67 and anti-mouse immunoglobulin G (IgG) antibodies for 2 h at room temperature. After washing in PBS, slides were incubated with an horseradish peroxidase (HRP)/Fab polymer conjugate for another 30 min. The sites of peroxidase activity were visualized using 3,3’-diamino-benzidine tetrahydrochloride as a substrate. Gill Hematoxylin Solution II (MERCK, Darmstadt, Germany) was utilized as the counterstain. All specimens were deparaffinized and stained with hematoxylin and eosin (H&E) which was used as a light counterstain.
Values represent the means ± standard deviation and the experiments were repeated at least three times. Statistical analyses were performed using the one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used when more than three groups were analyzed. Data comparisons were performed with Student’s t test (Sigma-Stat 2.0, Jandel Scientific, San Rafael, CA) when two groups were compared. In all cases, a p value <0.05 was considered to be statistically significant.
This study was supported by grants from National Science Council, Taiwan (MOST 103-2314-B-371-007-MY2) (MOST 104-2314-B-371-008-MY2) and Changhua Christian Hospital (104-CCH-MST-006) (104-CCH-MST-009) (104-CCH-IRP-094) (104-CCH-IRP-061) (103-CCH-ICO-006). The authors of the manuscript do not have a direct financial relation with the commercial identity mentioned in this paper.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015; 65:87-108.
2. Bhattacharyya N. The impact of race on survival in nasopharyngeal carcinoma: a matched analysis. Am J Otolaryngol. 2004; 25:94-97.
3. Gondhowiardjo S. Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) and tumor proliferation rate as predictive factors of nasopharyngeal cancer (NPC) radiation response. Gan to kagaku ryoho. 2000; 27:323-331.
4. Maiuri MC, Zalckvar E, Kimchi A and Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007; 8:741-752.
5. Eisenberg-Lerner A, Bialik S, Simon HU and Kimchi A. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell death and differentiation. 2009; 16:966-975.
6. Lockshin RA and Zakeri Z. Apoptosis, autophagy, and more. The international journal of biochemistry & cell biology. 2004; 36:2405-2419.
7. Young MM, Kester M and Wang HG. Sphingolipids: regulators of crosstalk between apoptosis and autophagy. J Lipid Res. 2013; 54:5-19.
8. Cohen D, Higman SM, Hsu SI and Horwitz SB. The involvement of a LINE-1 element in a DNA rearrangement upstream of the mdr1a gene in a taxol multidrug-resistant murine cell line. The Journal of biological chemistry. 1992; 267:20248-20254.
9. Davies AM, Lara PN, Jr., Mack PC and Gandara DR. Docetaxel in non-small cell lung cancer: a review. Expert opinion on pharmacotherapy. 2003; 4:553-565.
10. Green MR. The evolving role of gemcitabine and pemetrexed (Alimta) in the management of patients with malignant mesothelioma. Clinical lung cancer. 2002; 3:S26-29.
11. Shacka JJ, Klocke BJ, Shibata M, Uchiyama Y, Datta G, Schmidt RE and Roth KA. Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Molecular pharmacology. 2006; 69:1125-1136.
12. Finlan LE, Kernohan NM, Thomson G, Beattie PE, Hupp TR and Ibbotson SH. Differential effects of 5-aminolaevulinic acid photodynamic therapy and psoralen + ultraviolet A therapy on p53 phosphorylation in normal human skin in vivo. The British journal of dermatology. 2005; 153:1001-1010.
13. Fitzpatrick FA and Wheeler R. The immunopharmacology of paclitaxel (Taxol), docetaxel (Taxotere), and related agents. International immunopharmacology. 2003; 3:1699-1714.
14. McGrogan BT, Gilmartin B, Carney DN and McCann A. Taxanes, microtubules and chemoresistant breast cancer. Biochimica et biophysica acta. 2008; 1785:96-132.
15. Wong RW and Rabie AB. Effect of psoralen on bone formation. Journal of orthopaedic research. 2011; 29:158-164.
16. Zhang XD, Wang Y, Wang Y, Zhang X, Han R, Wu JC, Liang ZQ, Gu ZL, Han F, Fukunaga K and Qin ZH. p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum. Autophagy. 2009; 5:339-350.
17. Hsieh MJ, Yang SF, Hsieh YS, Chen TY and Chiou HL. Autophagy inhibition enhances apoptosis induced by dioscin in huh7 cells. Evid Based Complement Alternat Med. 2012; 2012:134512.
18. Chen PN, Hsieh YS, Chiou HL and Chu SC. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chemico-biological interactions. 2005; 156:141-150.
19. Huang HP, Shih YW, Chang YC, Hung CN and Wang CJ. Chemoinhibitory effect of mulberry anthocyanins on melanoma metastasis involved in the Ras/PI3K pathway. Journal of agricultural and food chemistry. 2008; 56:9286-9293.
20. Vieira JR, de Souza IA, do Nascimento SC and Leite SP. Indigofera suffruticosa: an alternative anticancer therapy. Evidence-based complementary and alternative medicine. 2007; 4:355-359.
21. Wang W, Guo J, Zhang J, Peng J, Liu T and Xin Z. Isolation, identification and antioxidant activity of bound phenolic compounds present in rice bran. Food Chem. 2015; 171:40-49.
22. Wang H, Zhai Z, Li N, Jin H, Chen J, Yuan S, Wang L, Zhang J, Li Y, Yun J, Fan J, Yi J and Ling R. Steroidal saponin of Trillium tschonoskii. Reverses multidrug resistance of hepatocellular carcinoma. Phytomedicine. 2013; 20:985-991.
23. Li ZH, Wan JY, Wang GQ, Zhao FG and Wen JH. Identification of compounds from Paris polyphylla (ChongLou) active against Dactylogyrus intermedius. Parasitology. 2013; 140:952-958.
24. Yin X, Qu C, Li Z, Zhai Y, Cao S, Lin L, Feng L, Yan L and Ni J. Simultaneous determination and pharmacokinetic study of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC-MS-MS. Biomed Chromatogr. 2013; 27:343-348.
25. Xiao XH, Yuan ZQ and Li GK. Separation and purification of steroidal saponins from Paris polyphylla by microwave-assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection. J Sep Sci. 2014; 37:635-641.
26. Zhang C, Jia X, Wang K, Bao J, Li P, Chen M, Wan JB, Su H, Mei Z and He C. Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells. PloS one. 2016; 11:e0147405.
27. He DX, Li GH, Gu XT, Zhang L, Mao AQ, Wei J, Liu DQ, Shi GY and Ma X. A new agent developed by biotransformation of polyphyllin VII inhibits chemoresistance in breast cancer. Oncotarget. 2016; 7:31814-24. doi: 10.18632/oncotarget.6674.
28. Lin Z, Liu Y, Li F, Wu J, Zhang G, Wang Y, Lu L and Liu Z. Anti-lung Cancer Effects of Polyphyllin VI and VII Potentially Correlate with Apoptosis In Vitro and In Vivo. Phytotherapy research. 2015; 29:1568-1576.
29. Zhang C, Jia X, Bao J, Chen S, Wang K, Zhang Y, Li P, Wan JB, Su H, Wang Y, Mei Z and He C. Polyphyllin VII induces apoptosis in HepG2 cells through ROS-mediated mitochondrial dysfunction and MAPK pathways. BMC complementary and alternative medicine. 2016; 16:58.
30. Kondo Y, Kanzawa T, Sawaya R and Kondo S. The role of autophagy in cancer development and response to therapy. Nature reviews Cancer. 2005; 5:726-734.
31. Yang ZJ, Chee CE, Huang S and Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther. 2011; 10:1533-1541.
32. Levine B and Yuan J. Autophagy in cell death: an innocent convict? The Journal of clinical investigation. 2005; 115:2679-2688.
33. Sun H, Wang Z and Yakisich JS. Natural products targeting autophagy via the PI3K/Akt/mTOR pathway as anticancer agents. Anticancer Agents Med Chem. 2013; 13:1048-1056.
34. Strozyk E and Kulms D. The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence. Int J Mol Sci. 2013; 14:15260-15285.
35. Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C and Pan H. p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 2014; 344:174-179.
36. Cagnol S and Chambard JC. ERK and cell death: mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence. FEBS J. 2010; 277:2-21.
37. Chen YS, He Y, Chen C, Zeng Y, Xue D, Wen FY, Wang L, Zhang H and Du JR. Growth inhibition by pennogenyl saponins from Rhizoma paridis on hepatoma xenografts in nude mice. Steroids. 2014; 83:39-44.
38. Kim AD, Kang KA, Kim HS, Kim DH, Choi YH, Lee SJ, Kim HS and Hyun JW. A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells. Cell death & disease. 2013; 4:e750.
39. Hsieh MJ, Tsai TL, Hsieh YS, Wang CJ and Chiou HL. Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines. Archives of toxicology. 2013; 87:1927-1937.
40. Ko CP, Lin CW, Chen MK, Yang SF, Chiou HL and Hsieh MJ. Pterostilbene induce autophagy on human oral cancer cells through modulation of Akt and mitogen-activated protein kinase pathway. Oral oncology. 2015; 51:593-601.
41. Kim A, Yim NH and Ma JY. Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells. BMC complementary and alternative medicine. 2013; 13:233.
42. Li Y, Sun Y, Fan L, Zhang F, Meng J, Han J, Guo X, Zhang D, Zhang R, Yue Z and Mei Q. Paris saponin VII inhibits growth of colorectal cancer cells through Ras signaling pathway. Biochemical pharmacology. 2014; 88:150-157.
43. Li Y, Fan L, Sun Y, Miao X, Zhang F, Meng J, Han J, Zhang D, Zhang R, Yue Z and Mei Q. Paris saponin VII from trillium tschonoskii reverses multidrug resistance of adriamycin-resistant MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis augmentation. Journal of ethnopharmacology. 2014; 154:728-734.
44. Zhang W, Zhang D, Ma X, Liu Z, Li F and Wu D. Paris saponin VII suppressed the growth of human cervical cancer Hela cells. European journal of medical research. 2014; 19:41.
45. Chen LC, Wang LJ, Tsang NM, Ojcius DM, Chen CC, Ouyang CN, Hsueh C, Liang Y, Chang KP, Chen CC and Chang YS. Tumour inflammasome-derived IL-1beta recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma. EMBO Mol Med. 2012; 4:1276-1293.
46. Hsieh MJ, Lin CW, Chiou HL, Yang SF and Chen MK. Dehydroandrographolide, an iNOS inhibitor, extracted from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells. Oncotarget. 2015; 6:30831-30849. doi: 10.18632/oncotarget.5036.
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text:
All businesses are required to keep accurate records for both taxation and VAT purposes. Good bookkeeping practices also benefit the business by helping you manage and control your finances, plan for future growth and ensure that your accounts are completed more efficiently, which should in turn avoid late filing penalties.
We can provide you a simple spread sheet designed especially for our clients to record business transactions
You can post or drop off all your paper work, bank statements etc and we will record everything and prepare :
- Management Accounts
- VAT Returns
- Payroll Records
- Year End accounts
- Tax Calculations

domain: economics


text:
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domain: entertainment


text:
The 415th Bombardment Group was originally used by the School of Applied Tactics before becoming a training unit.
The 415th was activated on 15 February 1943 and was equipped with a mix of aircraft. It was used as a demonstration group by the Army Air Force School of Applied Tactics (at Orlando).
By March 1944 the group became a standard replacement training unit, but this was a short-lived assignment and it was disbanded on 5 April 1944.
Aircraft: Douglas A-20 Havoc, Douglas A-24 Banshee, Douglas A-26 Invader, North American B-25 Mitchell, Bell P-39 Airacobra
|12 February 1943||Constituted as 415th Bombardment Group (Dive)|
|15 February 1943||Activated|
|5 April 1944||Disbanded|
2d. Lt Michael J Panek: 1 Mar 1943
Maj Wesley E Dickerson: 12 Mar 1943
Lt Col Robert K Martin: 29 Mar 1943
Col John R Kelly: 23 Oct 1943
Lt Col Steele R Patterson: 6 Mar-5 Apr 1944
Alachua AAFM, Fla: 15 Feb
Orlando AB, Fla: 25 Feb 1944
Dalhart AAFM, Tex: 19 Mar-5 Apr 1944
465th Bombardment Squadron: 1943-44
521st (formerly 667th) Bombardment Squadron: 1943-44

domain: history


text:
Yes, you read that correctly.
Chocolate brownies + Kit Kats = PERFECTION 😍
Kit Kat Brownies
1 stick unsalted butter, cut into 1 inch cubes
3/4 – 1 cup brown sugar
3/4 cup cacao powder
1/2 tsp salt
1 tsp vanilla extract
2 large eggs
1/3 cup all-purpose flour
16 Kit Kats
1) Preheat oven to 325F. Line a 8x8x2 baking pan & set dish aside.
2) Melt butter in a small sauce-pan over medium heat. Mix sugar, cacao, and salt in a medium bowl. Pour butter in a steady stream into dry ingredients, whisking constantly to blend.
3) Whisk in vanilla & eggs one at a time, beating vigorously to blend after each addition.
4) Add flour and stir until just combined (do not overmix).
5) Pour half the batter into the pan, then neatly place Kit Kats on top. Pour the remaining batter over the layer of Kit Kats.
6) Bake for 25-30 minutes. Keep a close 👀 on the brownies! Make sure they don’t burn!! (That would be depressing)
Makes 16 brownies. ENJOY! 😘
Make sure we stay connected on…
Snapchat: @medhacooks to see the behind the scenes of my cooking experiments
Instagram: https://instagram.com/medhacooks/ To see the final pictures of my creations

domain: food


text:
MEXICO CITY — According to Reuters, the Mexican front-runner for next year’s election, Andres Manuel Lopez Obrador, formalized his bid for the presidency on Tuesday Dec. 12 and promised his government would spend on the young, elderly and farmers.
Left-winger Lopez Obrador, who had a 12-point lead in one recent poll, wants to significantly change Mexico’s approach to the economy, security and education, vowing more support for the poorest but without new taxes or higher debt levels.
He promised cheap fertilizer and fixed produce prices for farmers with a goal of making Mexico self-sufficient in food. He also offered paid apprenticeships for unemployed youth, grants for students and higher pensions for the elderly — expanding on popular welfare programs introduced when he governed Mexico City.
A win by the 64-year-old self-declared nationalist on July 1 could reverse a Latin American trend toward right-leaning governments and set the stage for friction with U.S. President Donald Trump over his anti-migrant language and policies.
Lopez Obrador promised friendly ties with the U.S. government but said he would not accept “racist, hegemonic or arrogant attitudes.”
He also proposed a shakeup of government, saying he would move more than a dozen ministries and federal bodies including state oil company Pemex from the capital to regional towns.
“The federal government will be decentralized,” Lopez Obrador said, in a speech in the capital after registering his intention to run for his National Regeneration Movement (MORENA) party. He also promised not to increase fuel prices.
In the clearest language yet, he repeated his intention to consult with victims of drug crime about the possibility of offering amnesty to criminals who commit to rehabilitation.
A poll this week found that two-thirds of Mexicans rejected the idea of amnesty, in a country on track for its deadliest year in modern history with nearly 21,000 murders through October.
Lopez Obrador, who has unsuccessfully run for the presidency twice before, will likely face Jose Antonio Meade, running for the ruling party, and Ricardo Anaya, who heads a left-right coalition of opposition parties.
He did not say how he would finance his spending plans, but in the past has said all new spending would be funded by ending government corruption and waste.

domain: news


BY TTT NEWS NETWORK
KOLKATA, 29 DECEMBER 2022
“Metro rail is another achievement of Bangladesh,” said Prime Minister Sheikh Hasina Wajed on Wednesday.
She said this while inaugurating a section of the 20 kilometres urban rail project known as Uttara Line 6, which is the the first ever metro railways in the capital city of Dhaka, one of the most populous and congested cities in the world.
She has also become a passenger of the train by riding from Diabari in Uttara to Agargaon. A total 285 people took ride in the first journey including , Sheikh Rehana, the younger sister of the PM and other ministers of her cabinet.
The metro railway line connects Dhaka’s Northern area to the centre of government offices and hospitals. It will eventually pass through the city to Motijheel’s financial sector in the South.
To mark this historic occasion, she has also unveiled a commemorative stamp and a banknote.
Every day the Bangladesh’s economy loses 3.2 million working hours due to traffic congestion which costs the country’s economy billions of dollars annually.
Only six months ago, PM Sheikh Hasina Wajed has inaugurated the longest bridge upon a river in Padma which has given a a giant leap forward in the growth of economy and tourism and has acted as game changer in the roadways infrastructure sector.
Dhaka Mass Transit Company Limited (DMTCL) said that a total of 200 passengers can board the train and it will run for four hours daily initially from 8 AM to 12 PM.
The current fare of the route will be Bangladesh Taka 60 and will take total ten minutes to reach the destinations. The trains will run after a durations of ten minutes each.
Six women drivers have been trained to run these 12 metro rails and on the inaguration day the women driver was Mariam Afeeja. Each train has six coaches.
Due to the outbreak of the covid -19 pandemic the project has been delayed.
The metro railways will have total 17 stations. The project has been funded by Japan International Cooperation Agency.
Construction began on 26 June 2016 with an inauguration ceremony presided over by the reigning prime minister Sheikh Hasina. Civil engineering work was done by the Italian-Thai Development Public Company Ltd. and China’s Sinohydro Corporation Ltd. JV, and a Tokyo-based construction company developed the depot’s land.
Once fully opeartonal the metro railways will be able to carry 60,000 passengers per hour.


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Transcription factor Stat5 (signal transducer and activator of transcription) is essential for PRL-induced terminal differentiation of mouse mammary epithelial cells during pregnancy and lactation and has been implicated in mammary tumorigenesis. A new and sensitive immunological method to detect active, tyrosine phosphorylated Stat5 in situ revealed that Stat5 is continuously activated in luminal epithelial cells of mouse and human breast, not only during pregnancy and lactation, but also outside of pregnancy. Examination of virgin Stat5a or Stat5b null mice suggested that Stat5a was the primary isoform activated in mammary epithelial cells. Basal activation of Stat5 in mammary epithelium of virgin wild-type mice was continuous throughout estrous cycle and was also detected in 17 of 17 normal human breast tissue specimens analyzed. PRL was identified as the principal factor maintaining basal activation of Stat5 in mammary epithelium of nonpregnant mice based on several lines of evidence. First, administration of PRL, but not GH or epidermal growth factor, uniformly enhanced basal activation of Stat5 in luminal mammary epithelial cells. Second, hypophysectomy disrupted basal activation of Stat5, an effect that was completely reversed by administration of PRL, but only partially by GH. Third, knock-out of the PRL receptor gene markedly reduced basal activation of Stat5, an effect that was maintained in a normalized endocrine environment after transplanting PRL receptor null mammary epithelium into wild-type mice. Continuous activation of Stat5 indicates a role of this transcription factor in normal, nonpregnant breast epithelial cells, and may shed new light on Stat5 involvement in breast tumor promotion.
ASJC Scopus subject areas
- Molecular Biology

domain: medical


A device that makes compost in 24 hours
by Lorenz Keller
Waste bins, also called rubbish bins, have become an indispensable part of our everyday lives. They are used to store, dispose of or separate waste and are therefore an important part of every household or workplace. There are different models to choose from depending on your needs and intended use.
A frequently encountered waste bin is the classic standing bin. This is usually made of plastic or metal and has a lid to keep odours and flies out. It is available in different sizes and designs and is suitable for use in kitchens, bathrooms or offices, depending on the size. The operation varies from manual variants to those with a practical touch sensor.
For use directly under the sink, there are so-called waste systems. This is a practical and space-saving solution for collecting and disposing of waste in the kitchen. These systems are particularly popular in kitchens where space is limited or a minimalist design is preferred. They consist of several bins that are fixed in a frame under the sink. The bins are usually made of plastic or stainless steel and have different sizes to accommodate different types of waste.
Another type of waste bins are waste separation systems, also called recyclables separation bins. Compared to the previously mentioned waste systems, waste separation systems have a much larger volume and do not fit under the sink. They also consist of several containers that are assigned to different types of waste. For example, paper, glass and plastic can be disposed of separately for optimal recycling.
There are special outdoor waste bins for use outdoors or in public areas. These are robust and weatherproof and can be mounted on walls as well as on stands. Their size and capacity make them ideal for use in playgrounds, parks or public spaces. Another advantage is that most outdoor litter bins have an integrated ashtray.
Small cosmetic bins are particularly suitable for use in bathrooms or guest toilets. These are usually made of plastic or stainless steel and have a lid to prevent unpleasant odours. They are available in various sizes and designs, such as a practical pedal bin, and can also be an eye-catcher visually.
The wastepaper bin is usually a relatively small and lightweight container made of plastic, metal or other materials. As the name suggests, it is mainly used in offices, schools, households and other working or living environments to enable the proper collection and disposal of paper waste. There are different designs and sizes of waste paper baskets that can be adapted to the specific needs of the place of use.
In summary, there is a wide variety of waste bins that can be selected according to need and intended use. There is a suitable model available for every need and place of use. When choosing, pay attention to the capacity, the materials and any additional functions that may be required, such as lids or fastening options.


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Columbus-based Cummins Inc. (NYSE: CMI) is making changes to its top leadership, the company announced Thursday. Tom Linebarger will step down as chief executive officer on August 1, though he will remain chairman of the board. Jennifer Rumsey, who currently serves as president and chief operating officer, will be promoted to president and CEO, becoming the first woman to lead the company in its more than 100-year history.
Linebarger will also serve as executive chairman once the change takes effect. Cummins is the state’s third-largest public company, according to IBJ research, based on 2021 revenue of $24 billion.
“Jen is a once in a generation talent and the right leader for Cummins at this important time in our history,” Linebarger said in written remarks. “Jen uniquely understands our customers and business, having worked in many different parts of the business during her more than 20-year career, and in every role, she has consistently delivered results.”
Rumsey joined Cummins as a technical advisor in 2000 and was named president and COO in March 2021 after serving as vice president and president of Cummins’ components segment. She also previously held the role of chief technical officer.
Rumsey was elected to the Cummins Board of Directors in February and the company says she will maintain her seat on the board.
“Growing up in Columbus, Indiana, where Cummins was founded, and spending most of my career here makes this announcement incredibly meaningful,” said Rumsey. “I am grateful to Tom for his support and mentorship over the years and to the Board for their confidence in my leadership. My technical background, business experience and focus on people, purpose, and impact have prepared me for this moment.”
Linebarger began his career at Cummins as an intern before joining the company full-time in 1993. He has held numerous executive positions at the company, including serving as president and COO from 2008 to 2011.
Cummins says in his role as executive chairman, Linebarger will work with Rumsey on specific initiatives, including the company’s planned $3.7 billion acquisition of Meritor Inc. (NYSE: MTOR) in Michigan. The acquisition is expected to close by the end of the year.
In a news release, Cummins Lead Director Tom Lynch thanked Linebarger for his work over the last three decades.
“His leadership has grown Cummins’ global business, positioned the company to lead in zero- and low-carbon solutions, and developed a highly capable and diverse management team to lead us through the technology transition,” said Lynch. “We look forward to his continued contributions as Executive Chairman.”
According to Cummins’ latest proxy statement, Linebarger realized more than $12 million in compensation in 2021, while Rumsey realized more than $2.6 million. In a public document released Thursday, Cummins said Linebarger’s base salary would be reduced to $1.15 million, while his target annual bonus would be increased to 150% of base salary.
Rumsey’s base salary was being increased to $1.5 million, with possible incentive bonuses topping $10 million.
The leadership change comes two months after Cummins shareholders rejected a proposal to split the duties of chairman and chief executive officer. At the time, the board said in a proxy statement that it believed “it is in the best interests of our company for the roles of our Chairman and Chief Executive Officer to be combined.”
Cummins did not provide a timeline for naming a successor to Rumsey as COO.
In a March 2021 interview on Inside INdiana Business with Gerry Dick, Rumsey discussed women executives operating in what has traditionally been male-dominated space and how she broke the glass ceiling.
You can watch the full interview by clicking here.


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With a stay at Hotel Vista Grande Osaka, you'll be centrally located in Osaka, steps from Dotombori Konamon Museum and Hozenji-Yokocho Alley. This 4-star hotel is close to Osaka Science Museum and Osaka Castle.
Make yourself at home in one of the 304 air-conditioned rooms featuring refrigerators and flat-screen televisions. Your Select Comfort bed comes with down comforters. Complimentary wired and wireless Internet access keeps you connected, and digital programming provides entertainment. Private bathrooms with shower/tub combinations feature deep soaking bathtubs and complimentary toiletries.
Enjoy a satisfying meal at a restaurant serving guests of Hotel Vista Grande Osaka. Breakfast is available daily for a fee.
Business, Other Amenities
Featured amenities include complimentary high-speed (wired) Internet access, a computer station, and dry cleaning/laundry services. Self parking (subject to charges) is available onsite.
Modern facilities Spider in the ice-bowl, poor breakfast select and over-priced
The location of the hotel, it's really in the entertainment area
Excellent location and room. If I ever be in Osaka again, I would be in this hotel.
Staffs not well in english communication and no wi-fi available.Only LAN cable provided.

domain: travel


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Life Science REIT makes maiden purchase
Life Science REIT, the recently launched real estate investment trust focused on U.K. life science properties, has completed its maiden acquisition with a £77 million (€90 million/$102 million) purchase in London.
The group, which raised £350 million (€410 million/$463 million) in an initial public offering (IPO) in November, has bought Rolling Stock Yard, a nine-story office and laboratory building near London’s St Pancras station from a joint venture of Newmark Properties, Argo Real Estate and Investec Bank.
Rolling Stock Yard, which comprises more than 50,000 square feet of office and laboratory space, is located on York Way just north of King’s Cross and St Pancras stations. The building was completed in March 2020.
Occupancy of the property, which has attracted major life science companies including Gyroscope Therapeutics, is at 76 percent.
Simon Farnsworth, managing director of Ironstone Asset Management, the company’s investment adviser


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McDonald’s partners with Deliveroo
McDonald’s and Deliveroo recently announced a new, long-term global strategic partnership. They say it will evolve their existing agreement and provide customers with the convenience and value of McDelivery through the Deliveroo platform in markets across the world.
The partnership includes the UK, Deliveroo’s largest market, where McDelivery will become available via the delivery company during the second quarter. It is expected to cover a number of McDonald’s UK restaurants initially when it begins in the summer, with expansion across the country planned for later in the year.
Alistair Macrow, McDonald's UK and Ireland CEO, said: “More than 1,200 of our restaurants now offer deliver, and this offer continues to be incredibly popular with customers. We look forward to welcoming Deliveroo and their riders into our suite of McDelivery partners, enabling us to deliver directly to more of our customers across the UK.”
Carlo Mocci, Deliveroo’s chief business officer UKI, added: “We are delighted to be expanding our partnership with McDonald’s through this global agreement. Our mission is to become the definitive online food company and working with McDonald’s will help us to achieve this.”

domain: geography


This conductivity sensor is built indoor or outdoor use. Two high grade steel probes mounted to a grounded twisted shielded pair with drain wire for signal insulation and use in close vicinity to pump control conductors.
Sensor is rated for low voltage DC use and conducts in the MilliAmp range when the probes touch clear water. The sensor is an open circuit when wet an performs as a 10K Ohm Resistor when in contact with water. The black conductor is the Anode, and white is the cathode.
The unit ships with 4 Flexible C-Clamps for mounting to 1" to 3.5" pipe, and (4) 10" Wire ties. There are no additional instructions in the shipment.


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New DNA evidence claims to unveil identity of Jack the Ripper
19 March, 2019, 21:52
After more than 130 years of mystery, researchers believe they might have finally discovered the true identity of one of the world's most notorious killers, Jack the Ripper.
Jack the Ripper committed five murders in London's Whitechapel district, and up to six more deaths have at times been attributed to the killer.
The evidence is a stained silk shawl found next to the mutilated body of one of the victims: Catherine Eddowes.
They wrote: "We describe for the first time systematic, molecular level analysis of the only surviving physical evidence linked to the Jack the Ripper murders". The shawl's provenance is unproven, but it is spattered with stains that appear to be blood and semen.
However, many researchers dispute Miller and Louhelainen's physical evidence, claiming there is no proof the shawl was ever present at Jack the Rippers crime scene.
Over a century later, scientists from Liverpool John Moore's university in the United Kingdom claim to have conducted the "most advanced study to date regarding this case". They pointed out that Louhelainen and Miller failed to mention the specific genetic variants they identified and compared between DNA samples.
Eddowes was the second woman to have died at the hands of Jack the Ripper that night - he had slit Elizabeth Stride's throat an hour earlier.
Cohen warrant materials have been released A judge ordered those sections to remain secret after prosecutors said they were still investigating campaign finance violations. Cohen has since pleaded guilty to campaign finance violations, which he says Trump directed, and other charges.
This prompted Reuters to conclude that the official disclosure of talks boosts the chances that both banks conclude the agreement. Deutsche Bank has a market value of some 16 billion euros ($18.1 billion), while Commerzbank weighs in at about 9 billion euros.
Given the differences between RTX and GTX cards, this means the ray tracing experience on them will not be equal though. At CES 2019, NVIDIA enabled VESA Adaptive-Sync technology available across all GeForce cards on non-G-SYNC panels.
The president accused the Democrats of trying to use impeachment to "steal" the US presidency away from him. Only 28.4 percent said they believed the House "should seriously consider impeaching President Trump".
The Samsung Galaxy A10 is the most affordable Samsung smartphone to come Android Pie operating system out-of-the-box. There is a USB Type-C port at the bottom alongside the speaker grille, 3.5mm audio jack, and a microphone.
Strive for at least 150 minutes a week of moderate-intensity exercise, such as brisk walking and swimming , the guidelines say. And according to doctor Deidre Mattina, a Henry Ford Hospital cardiologist.
Born in 1940, Mr Nazarbayev came to power as first secretary of the Communist Party of Kazakhstan when it was a Soviet republic. Kazakhstan President Nursultan Nazarbayev announced his shock resignation on Tuesday, March 19, 29 years after taking office.
Indonesia is located on the geologically active Pacific Ring of Fire and regularly experiences earthquakes and volcanic activity. The landslides trapped a total of 35 visitors including Malaysian tourists and all of them have been rescued safely.
In February, authorities announced that Kraft was videotaped twice paying for a sex act at an illicit massage parlor. Kraft was among hundreds of men charged for soliciting prostitution in Florida last month.
Pujols agreed to a $240 million, 10-year deal before the 2012 season but has not matched his previous performance with St. So Betts very well may be looking at doubling that $200 million offer when he comes to free agency after the 2020 season.
Arsenal are looking for a replacement for Sven Mislintat , who left the Gunners as head of recruitment in February. Marc Overmars has extended his contract as Ajax Director of Football until June 2024.
Christchurch Shootings: Brenton Tarrant Appears in Court
He with his two sons when the shooter opened fire during Friday prayers. "Just helping people is his main thing". Syrian refugee Khaled Mustafa was killed in the attack, Syrian Solidarity New Zealand said on its Facebook page.
Mugshot of Mama June Released After Crack Cocaine Arrest
June "Mama June" Shannon and her boyfriend, Geno Doak , were arrested on drug charges in Macon County, Ala. on March 13, 2019. Mama June and her boyfriend, Geno Doak , were arrested last Wednesday for the alleged possession of crack cocaine.
Warren supports elimination of electoral college
There are already efforts at the state level to diminish the effectiveness of the Electoral College in favor of the popular vote. The bill, which needs states representing at least 270 Electoral College votes to sign on in order to take effect, has 181.
Apple gives the iMac a performance boost
Following the iPad Air and iPad Mini , Apple has updated iMac desktops, with new Intel processors and AMD graphics . A second off-the-shelf configuration includes a Core i5 processor running at 3.0GHz and Radeon Pro 560X graphics.
Three teens dead after St Patrick's Day party in Tyrone
In that crush people seem to have fallen, we are examining CCTV footage to see if the people who had fallen were the deceased. Lauren Bullock, 17, Morgan Barnard, 17, and 16-year-old Connor Currie were all killed in the crush.
Fox News Finds Way to Attack AOC After Christchurch Massacre
An avowed white supremacist took credit for attacking two mosques in the city of Christchurch that were packed for Friday prayers. NRA spokeswoman Dana Loesch got involved, accusing Ocasio-Cortez, who was raised Catholic, of "mocking" prayer.
A 1,500 Ton Meteor Exploded Over Earth Last Year
The meteor entered the Earth's atmosphere nearly vertically at a steep angle of 7 degrees and at a speed of around 32km/s. But it went largely unnoticed until now because it blew up over the Bering Sea , off Russia's Kamchatka Peninsula .


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3DOM Studios is a creative independently owned 3D Animation and Visual Effects Company specialising in film and video production. We are based in the capital of Germany – Berlin. Based on our expertise and many years of experience, we help you to tell great stories and visualise your ideas without any limitations.
Known for our quality, efficiency and timely deliveries, our team of highly skilled professionals is ready to take on your creative project and turn it into reality.
Let’s get started
It’s never too early to start talking to us about your idea or project.
Looking for a production partner?
We are always open to interesting projects.

domain: artistic


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In an age of sex-texting and seemingly random sex among some teens, are Judy Blume’s books still relevant? Do they still reflect the teenage experience and what adolescents can expect in their middle and high school years? Are there still lessons to learn from these books written almost four decades ago?
For my generation growing up in the 1970s and 1980s, Blume’s books were the guideposts. They gave great insight about how to handle your first spin-the-bottle game, start your period or buy your first bra. I read almost all of Judy Blume’s books between elementary and middle school. Scenes and lines from the books are still vivid to me. I even remember the brand of maxi pads Margaret buys in “Are You There God? It’s Me, Margaret.” (Teenage Softies.)
My husband recently brought me home a book of essays written by women authors who had also grown up reading Blume books. In the book, “Everything I Needed to Know About Being a Girl I Learned from Judy Blume,” the women discuss how Blume influenced their perceptions of their bodies, boyfriends and friendships.
So I spent about two weeks re-reading many of the most popular Blume books specifically looking for how these books could relate to today’s world and teens. (I was encouraged when several of the books I needed had a wait list at my local library. I suspect that maybe those high school girls weren’t reading Blume but the middle school girls were. ) Here are my observations on Blume’s relevance today:
“Blubber” – These were the original “Mean Girls.” Blume didn’t have a name for it then but Wendy is the Queen Bee and Jill and Tracy were drones following along. I remember the main character Jill as being sympathetic, but she’s not at all. She’s just mean. I think Jill’s class would be arrested today for physically assaulting Blubber in the bathroom, and the school would be sued. As a parent I would be aghast if my child ever did anything like these girls did to another student. Although the forms of hazing and abuse have changed among teens (think email, texts and MySpace), I think the book could still help middle school students learn how deal with being bullied.
“Deenie” - I remember this book being entirely about masturbation. Oddly as I re-read it, I think I only counted two or three references to it in the entire book. Her main point on the masturbation is that it is normal and OK. I suspect teens still worry about masturbation today. Blume knows she’s a little bit dated on the Scoliosis info and has an editor’s note at the beginning of the new editions directing readers to a Web site for more information. I think the other big point in this book is teaching teens to feel good about themselves even if they have to wear a back brace, have pimples, have a small chest, etc.. Learning to love yourself is especially relevant in today’s society where low self esteems seems so rampant among our young women.
“Are you there God? It’s Me, Margaret” – Probably my favorite among all the Blume books, I think this one still really rings true. The settings don’t seem dated, and I think girls are still extremely concerned about their breasts developing and starting their periods. I’m wondering if this text was actually updated because I swear I remember them using a belt for the maxi pad and not sticky strips like are used today. (Wikipedia says they did update in 2006.) I think fourth and fifth graders would still find this book relevant. (This was one with a wait list at our local library so somebody is reading it.)
“Then Again, Maybe I won’t” – This was the boy book in the group. You may not have read it unless you had a brother. The boy’s father hits it big with an invention and this working-class, nice Italian family moves to a big house with a maid. The boy is obsessed with his neighbor’s sister and watches her undress at night. The big teen theme in this book is dealing with nocturnal emissions, which I’m sure both genders are still curious about.
However, I think one of the smaller themes in this book is actually more relevant today. The book examines how privilege can mess up kids’ values. The working-class boy is stunned by how unappreciative and lazy these affluent kids are. This book might be an eye-opener to some of our teens today.
“Forever” - The Biggie!! I did not read “Forever” – the infamous book where the teens actually do it – as a teen. I remember being offered the book in the locker room of my middle school. I was curious but was way too chicken to borrow it and take it home. I was certain my mother would know what the book was about as soon as she saw the title. I apparently gave my mother more credit than she deserved. She told me recently she didn’t even know “Are you There God? It’s Me, Margaret” was about getting your period.” (Quote from my mother: “I wondered why you never asked me any questions about periods.”)
For those of you who were certain that your mom would find out too and never read it, here is the gist of the plot: Senior high school girl meets senior high school boy at a FONDUE party. (OK that part is dated.) They fall madly in love and eventually after several months go all the way. The girl’s grandmother sends her literature about getting on birth control, and she goes to a Planned Parenthood clinic to go on the Pill. (Blume has a note to readers in the front of the book that with the advent of AIDS, condoms would be a better choice.)
The Equal Rights Amendment grandmother is a little dated and the fact the couple can get legally drunk at 18 will also seem odd to today’s teens.
At the time, this book was so scandalous because they had sex, but, hey, at least they thought they were in love. At least they were in a relationship. This reads more like a college relationship now. High school kids seem to have sex so randomly now. Maybe the book would help a teen realize that if they’re going to do it (which I’m definitely not saying they should), they should at least think they are in love and not just do it because they are bored or because they have low self esteem and are seeking acceptance.
I think this book would help teen girls understand why they are feeling so strongly about a boy and that those feelings might change even if they don’t think they ever will. I think this book would help explain a lot to young women about the sex act and the emotional responsibilities it brings, but it’s so real I hate to believe they would need that knowledge in high school. (But I guess some do.)
What do you remember about the Judy Blume books? Did you read “Forever” as a teen? Did your teens read them? Did they relate? Do you think they are still relevant today? Do kids just start reading them younger in our jaded world?

domain: library


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3 Website Design Trends Happening in 2019
As we head into 2019 I scoured the internet and talked with our team to find out what types of website design trends we should expect to hit websites this year. Regardless of the predicted trends and opinions I found, there is one common thread, one thing I think we can all count on – websites are and will continue to be a direct reflection of cultural trends, societal changes, and technological advancements we see in our world. Websites should complement the people that are using them.
Here are 3 ways websites will be keeping up with us users in 2019:
Clean up my clutter
Our lives are filled with clutter. Thanks to advancing technology, we literally have everything at our fingertips, not to mention the push to do and be everything. We are spread thin, overwhelmed, and all-out messy. Self-help books are now focused on saying “no,” decluttering your life, and finding breathing room. Well, website design in 2019 will be following suit.
Cluttered websites are a thing of the past. In 2019 you will see minimalism, clean sites, white space, and less copy, with more focus on what you need to know (nothing more, nothing less). White space (or any space that is devoid of copy and imagery) in web design is easier to read and navigate and allows for the user to directly focus on the most important elements of the page. This trend by no means is new but with this constant state of clutter, this trend will become even more of a focus this year as a way to create better user experiences and drive engagement.
Get my attention
We are bombarded by anywhere from 4,000-10,000 unique messages daily. Websites must continue to fight to get users’ attention. In 2019, expect that to come to life in the form of color and reimagined header areas. Websites are naturally built for bright colors and with the advancement of computer and mobile device screen resolutions, vibrant, saturated color is more possible and available to more users now than ever before. Color is one of the first things our brains perceive from a brand, so it’s often the first thing that pulls us in.
Hero or header areas are the top part of your webpage and the first thing people see when they land on your website. Needless to say, this area is VERY important not just for drawing someone’s attention but for encouraging engagement. Over the last couple of years, there has been a ton of experimentation with this area of websites to better reach visitors. I have no doubt we will continue to see changes in these spaces above the fold to include things like integrated video, custom illustrations and animation in 2019.
Keep up with me
There’s no doubt we are always on the go and good thing we have everything at our fingertips because we wouldn’t have time for anything less. From next-day delivery to AI doing the work for us, we need things that can keep up with how fast-paced we are and our surrounding world is. Websites have to keep up as well. Everything we do now starts on mobile and if your website isn’t ready for users on that device you are missing out A LOT. I see 2019 as the year where we not only continue to focus on mobile friendliness but in fact take it even further. Websites will be tailored to how we hold our phone with things like navigation and buttons at thumb’s length.
Site speed will also be a continued focus for websites in 2019 and there’s no reason why we should stop optimizing. One second delay in our hyper-speed world could mean exponential losses in users. You can test your website’s mobile speed now to see how many users you might be losing with Google Page Speed Insights.
2019 will be the year to focus on the needs of our users and meet them where they are with simple, minimalist design, attention-grabbing color and graphics, and efficient, easy-to-use websites. In 2019, you have an opportunity to connect with your customers authentically on the web. Start now!
Are you inspired or intrigued by these website design trends? Check out some of our recent work or email me! I’d love to chat about how we can use your website to better connect with your customers in 2019.

domain: design


Fri, Feb 22, 2013
Dreamliners Could Be Flying Again In Two Months, Sources Say
Boeing has an appointment with FAA Administrator Michael Huerta Friday to discuss a possible fix for the battery issues that have kept its Dreamliner airplanes grounded for more than a month. Sources close to the company say that if they are approved, the fleet could be carrying passengers again in two months.
The Associated Press reports that the planemaker believes that adding ceramic insulation between the battery cells and other changes, including a modified venting system, would prevent the batteries from overheating and causing a fire. It is not expected that Huerta will immediately give approval to the re-design, but at least it would be a starting point for a discussion of what standards Boeing will need to meet as it designs and tests the repair before installing it on the affected planes.
But the fix does not get to the root cause of why the batteries overheated in the first place, which has not yet been determined. Boeing's hope is that by proposing a safer design they can get the airplanes back in the air, and start to make inroads on a delivery backlog that is eating away at its cash reserves.
The proposed repairs would also include increasing the spacing between the battery cells, and modifications in the way they are installed in the airplane to reduce the effects of moisture, vibration, or swelling of the cells. It would also include a system to vent any hazardous material to the outside of the airplane, and monitor individual cells rather than the system as a whole.
Boeing thinks it could rebuild the battery systems on planes already in service by the end of March, but there is no guarantee that the FAA will approve the changes that quickly.
(Dreamliner image from file)
Next Step: Maiden Flight In First Quarter 2015 Dassault Aviation on Wednesday rolled back the curtains on the ultra long range Falcon 8X, the company's new flagship and the latest >[...]
Some Of Its Aircraft Are On Loan From Company Reorganizing Under Bankruptcy Laws The Evergreen Aviation and Space Museum in McMinnville, OR says that it is doing just fine, thank y>[...]
Also: Comet Water Differences, Garmin Datalink, Bogert Battery Boxes, WSI/iOs, Mistletoe Copter Fail Airbus hasn't seen a single airline order for its double-decker A380 superjumbo>[...]
Six Fatally Injured When Plane Impacted A Home The NTSB had released its preliminary report from an accident which occurred December 8, 2014 involving an Embraer EMB-500 Phenom 100>[...]
Also: $1M Aero-Photo, Draken Gets A-4s, 'Super Dell' Acts Super Dumb, Legendary Bell 47, Osprey Hours Evergreen Vintage Aircraft, LLC, the company that owns many of the aircraft on>[...]


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Just two days after hitting Netflix, the new Rian Johnson movie debuted Glass Onion: Takes the mystery out of the cutlery It dominates the Netflix Top 10 It is currently the #1 movie on the streaming device. Official viewing numbers for the movie won’t be released until Tuesday. But the big debut appears in the papers of the Murder-Mystery Squad. Perhaps the least surprising thing about the top 10 movies on Netflix right now is that five out of ten titles are Christmas movies, and only a few are Netflix originals. The most surprising thing about the top ten is what they toss in the butts glass onionis none other than the 2022 horror movie from Sony the invitation.
Johnson’s new film taking the top spot isn’t much of a surprise. the original Take out the knives Starring Daniel Craig, it grossed over $300 million at the worldwide box office and earned its way toward an Oscar nomination for Best Original Screenplay. Netflix quickly moved to pick up the movie’s sequel, Capturing the Glass Onion and the upcoming third “Knives Out / Benoit Blanc” mystery that will also be released on the streaming service. In past interviews, Johnson has joked that he’s eager to keep exploring the franchise with new mysteries and new murders for as long as audiences are interested in watching, and they clearly are. You can find the entire Netflix Top 10 below.
I still have to start writing knives out 3“,” Johnson told ComicBook.com in an interview about the movie. “I have to know what the next Benoit Blanc mystery is, what it’s called, where it’s set. So, I have a lot of work to do. So, TBD, I don’t want to give myself a deadline yet because I definitely won’t meet him.”
Read more glass onion content here:
#9 – Volcano: Rescue from Whakaari
#6 – Christmas Chronicles 2
#2 – Calling
#1 – Onion Glass: Knives Out Mystery
“Infuriatingly humble web fan. Writer. Alcohol geek. Passionate explorer. Evil problem solver. Incurable zombie expert.”
Katy Perry reveals she’s been sober for five weeks…and even made a pact with fiancé Orlando Bloom
Detective Who Unveiled Gwyneth Paltrow’s Ski Bag Group Chat: “Comically Easy”
New York Times Crossword Answers: The Jennings Actor Who Won The Best Actor Oscar


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Gucci Mane No Luv Mp3
Gucci Mane feat. Key Glo Pulls through with a new song titled “No Luv ” and it right here on Beatspice for your fast download.
Listen & Download Gucci Mane – No Luv feat. Key Glo Below:
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