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Piperlongumine selectively kills hepatocellular carcinoma cells and preferentially inhibits their invasion via ROS-ER-MAPKs-CHOP
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Yong Chen1, Ju Mei Liu1, Xin Xin Xiong1, Xin Yao Qiu1, Feng Pan2, Di Liu2, Shu Jue Lan3, Si Jin4, Shang Bin Yu1 and Xiao Qian Chen1
1 Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Institute of Brain Research, Key Laboratory of Neurological Diseases, Ministry of Education, Hubei Provincial Key Laboratory of Neurological Diseases, Huazhong University of Science and Technology, Wuhan, China
2 Department of Urology, Union Hospital, Huazhong University of Science and Technology, Wuhan, China
3 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
4 Department of Pharmacology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China
Xiao Qian Chen, email:
Keywords: piperlongumine, hepatocellular carcinoma, oxidative stress, signaling pathway, chemotherapy
Received: December 21, 2014 Accepted: January 21, 2015 Published: January 31, 2015
Hepatocellular carcinomas (HCC) are highly malignant and aggressive tumors lack of effective therapeutic drugs. Piperlongumine (PL), a natural product isolated from longer pepper plants, is recently identified as a potent cytotoxic compound highly selective to cancer cells. Here, we reported that PL specifically suppressed HCC cell migration/invasion via endoplasmic reticulum (ER)-MAPKs-CHOP signaling pathway. PL selectively killed HCC cells but not normal hepatocytes with an IC50 of 10-20 µM while PL at much lower concentrations only suppressed HCC cell migration/invasion. PL selectively elevated reactive oxygen species (ROS) in HCC cells, which activated or up-regulated downstream PERK/Ire 1α/Grp78, p38/JNK/Erk and CHOP subsequently. Administration of antioxidants completely abolished PL’s effects on cell death and migration/invasion. However, pharmacological inhibition of ER stress-responses or MAPKs signaling pathways with corresponding specific inhibitors only reversed PL’s effect on cell migration/invasion but not on cell death. Consistently, knocking-down of CHOP by RNA interference only reversed PL-suppressed HCC cell migration. Finally, PL significantly suppressed HCC development and activated the ER-MAPKs-CHOP signaling pathway in HCC xenografts in vivo. Taken together, PL selectively killed HCC cells and preferentially inhibited HCC cell migration/invasion via ROS-ER-MAPKs-CHOP axis, suggesting a novel therapeutic strategy for the highly malignant and aggressive HCC clinically.
Liver cancers are highly malignant and aggressive tumors, making it the third leading cause of cancer death in the world . Hepatocellular carcinoma (HCC) is the most common type of liver malignant tumors in adults. HCC usually occurs following a viral hepatitis infection (hepatitis B or C) or cirrhosis, in which complicated pathological processes induced by reactive oxygen species (ROS) are heavily involved . ROS is considered as a two-edge sword in tumorigenesis and development. At the initial stage in HCC development, ROS or oxidative stress-responses mainly stimulate hepatocarcinogenesis or promote HCC development by activating pro-oncogenic gene activities or oncogenic signaling pathways such as mitogen-activated protein kinases (MAPKs) [3, 4]. Along with the progression of HCC, ROS overproduction or long-lasting oxidative stress-responses would tend to induce cell death or prevent cancer development in HCC [1, 5]. Therefore, modulating homeostasis of ROS or oxidative stress-responses has become an important therapeutic strategy for HCC [5, 6]. Accumulative evidences support that oxidants have therapeutic effects in cancer treatment and the underlying mechanisms have been well studied [5, 7-11]. It is well-known that ROS may alter various cellular signaling pathways profoundly depending on different cellular contexts and MAPKs (i.e., p38, JNK and Erk) are canonical ROS-responsive signaling pathways [8-12].Until now, the exact mechanisms of oxidative stress-responses in HCC development and treatment remain far from clear.
Piperlongumine (PL) is a biological active alkaloid existing largely in the long pepper (Piper longum L). PL has been traditionally used for treating gastrointestinal and respiratory diseases in Ayurvedic medicine . Recently, PL was identified as a highly reliable and potent cytotoxic compound in killing cancer cells in screening study . PL selectively kills cancer cells but leave normal cell intact as PL induces ROS accumulation only in cancer cells [8, 9, 13]. The PL induced selective accumulation of ROS in cancer cells represents a novel therapeutic strategy for cancers [8, 9, 13, 14]. It is reported that PL might exert its cytotoxicity by activating p38 [9,11], JNK , Erk , Akt [16, 17], promoting protein glutathionylation , or suppressing NFκB activities  in different types of cancer cells. Further exploring the anticancer effects as well as its underlying mechanisms of PL is required for its clinical applications.
Endoplasmic reticulum (ER), a specific organelle for Ca2+ storage and proper protein folding/maturation, plays an important role in regulating ROS homeostasis and stress-responses . Upon various pathological stimuli such as ROS or misfolded/unfolded proteins accumulation, ER homeostasis is disturbed and ER stress-responses are induced, leading to the activation of various downstream signaling pathways such as MAPKs and the induction of C/EBP homologous protein (CHOP) [21, 22]. Consequently, stressed cells may either restore its homeostasis or undergo programmed cell death such as apoptosis or autophage . In various cancer cells including HCC cells, enhanced ER stress-responses have been well documented [24-26]. However, the effects of ER stress-responses (either promoting or inhibiting cancer development) vary depending on specific ER-downstream signaling pathways in specific cellular contexts [24, 27]. Considering the central role of ER in oxidative stress-responses in HCC, it is likely that ER-mediated stress-responses and its downstream signaling pathways might be heavily involved in PL’s biological effects in HCC cells.
In the present study, we examined the anticancer effects of PL on HCC cells in vitro and in vivo, and further investigated the underlying mechanisms. Our data demonstrated that PL preferentially suppressed HCC cell migration and invasion via ER-MAPKs-CHOP depending on ROS.
Piperlongumine selectively kills HCC cells but not normal hepatocytes
We tested the cytotoxic effects of PL in human HCC cell lines to evaluate the therapeutic potential of PL for HCC. MTT assays demonstrated that 24 h of PL treatment induced a significant reduction in the cell viability of various HCC cells (HepG2, Huh7, LM3) in a concentration-dependent manner (PL 0, 5, 10, 15 and 20 µM), but had little effect in that of primary rat hepatocytes and normal hepatic L-02 cells (Fig. 1A). The half maximal inhibitory concentration (IC50) of PL in HCC cells was in the range of 10-20 µΜ. Trypan blue assay showed that the rate of cell death was significantly increased in HepG2, Huh7 and LM3 cells at 24 h of PL treatment in a dose-dependent manner (5-20 µM) (Fig. 1B). Consistently, the rate of cell death as revealed by propidium iodide (PI) staining (PI-positive nuclei) was evidently increased in HepG2, LM3 (Fig. 1C) and Huh7 (Supplemental Figure 1) cells at 24 h of PL treatment in a dose-dependent manner (5-20 µM). Further, the number of apoptotic cells as revealed by annexin V staining (annexin V-positive cells) was significantly increased at 24 h of PL-treatment (20 µM) in HepG2 cells (Fig. 1D). In addition, flow cytometry (FCM) analysis showed that at 24 h of PL-treatment (20 µM), the percent of sub-G1 cells (representing apoptotic cells) was increased in HepG2 cells while the ratio of G1/(S+G2) was not altered (Fig. 1E). Taken together, PL selectively and effectively induced cell death including apoptosis in HCC cells.
Figure 1: Piperlongumine selectively killed HCC cells but not normal hepatocytes. (A) Results of MTT assays showed the selective cytotoxicity of PL in HCC cells. HCC cell lines (HepG2, Huh7 and LM3), normal hepatic cell line (L-02) and primary rat hepatocytes were grown in 96-well plates for 24 h and subjected to PL treatment (0, 5, 10, 15 or 20 µM) for 24 h. Cell viability was measured by MTT assays (n=3). All values represented mean ± SEM of three independent experiments. (B) Statistical analysis of cell death in PL-treated HCC cells or primary hepatocytes as measured by trypan blue exclusion assays. *P<0.05, **P<0.01 (n=3). (C) Representative micrographs of PI staining showed cell death in PL-treated HepG2 and LM3 cells. Bar, 20 µm. (D) Representative results of FCM analysis showed apoptosis in PL-treated HepG2 cell. HepG2 cells were treated with PL (0, 10 or 20 µM) for 24 h. After annexin V-PI double staining, cells were subjected to FCM analysis. The percentages of cells in the Q2 together with Q4 quarters were calculated as apoptotic cells for statistical analysis in the right panel. **P<0.01 vs. PL 0 µM (n=3). (E) Representative results of FCM analysis showed the effects of PL in cell cycle of HepG2 cells. HepG2 cells were treated with 20 µM PL for 24 h. After PI staining, cells were subjected for FCM analysis. The arrow indicated the sub-G1 population.
Piperlongumine preferentially suppresses HCC cell migration and invasion in vitro
HCC are highly malignant tumors and cancer cell invasion or metastasis is the major cause of death. We examined the effects of PL on the ability of HCC cell migration and invasion in vitro. In the scratch-wound healing assay, cells along the wound-edge would migrate into the nude space after scratching and the scratch-wound would heal continuously (represented by the reduced scratch-wound sizes between the opposite scratching lines). In normal hepatic L-02 cells, PL treatment (within 10 µM) had no evident effect on the migration and the wound-healing speed was not altered within 48 h of PL treatment (Fig. 2A). In Huh7 (Fig. 2B) and HepG2 (Fig. 2C), however, PL prominently inhibited HCC cell migration and reduced wound-healing speed at 24 or 48 h of PL treatment in a dosage-dependent manner within 2.5-10 µM. Statistical analysis demonstrated that PL at lower concentrations (2.5, 5 or 10 µM) significantly reduced wound sizes of Huh7 (Fig. 2B) or HepG2 (Fig. 2C) cells while cell death in HepG2 or Huh7 cells was not altered at 48 h of PL treatment at 10 µM (Supplemental Fig. 2). Consistently, PL (10 µM) prominently reduced HepG2 cell migration at 48 h of PL treatment in the transwell migration assays (left upper panels, Fig. 2D). Further, PL (10 µM) significantly reduced invaded HepG2 cells in the transwell invasion assay at 48 h after PL-treatment (left lower panels, Fig. 2D). Statistical analysis demonstrated that migrated or invaded HepG2 cells were significantly reduced at 48 h of PL treatment (10 µM) (right panel, Fig. 2D). These evidences together demonstrated that PL preferentially suppressed HCC cell migration/invasion at lower PL concentrations.
Figure. 2: Piperlongumine selectively suppressed HCC cell migration or invasion in vitro. (A) Representative micrographs showed the effects of PL on L-02 cell migration after scratching. Confluent L-02 cells (normal hepatic cell line) in 35-mm culture dishes were subject to cell scratching with yellow pipettes. PL was added to the cultures with fresh culture media after brief culture rinsing. The scratch-wounds were photographed at various time points (0, 24 and 48 h) after cell scratching and the wound sizes were measured. Bar, 100 µm. Statistical analyses (right panel) demonstrated that PL with 10 µM did not alter L-02 cell migration significantly (n=3). (B-C) Representative micrographs showed the effects of PL on Huh7 (B) and HepG2 (C) cell migration after cell scratching. Bar, 100 µm. Statistical analyses (right panel) demonstrated that PL with 2.5-10 µM significantly altered Huh7 cell migration 48 h after cell scratching (n=3). *P<0.05, **P<0.01 vs. corresponding PL 0 µM control (n=3). (D) Representative micrographs showed the effects of PL on HepG2 cell migration and invasion. HepG2 cells were seeded into the upper chamber of transwell apparatus without (upper panel) or with (lower panels) matrigel. Drugs (PL alone or together with NAC or 4-PBA) were added to the culture 24 h after cell seeding. Cell migration (upper panels) and invasion (lower panels) were induced by FBS-containing media in the lower chamber. Migrated and invaded cells in the lower surface of the filters were stained and microphotographed 24 h after serum induction. Bar, 20 µm. Statistical analyses (right panel) demonstrated migrated or invaded HepG2 cells were significantly reduced upon PL treatment while co-treatment of NAC or 4-PBA significantly reversed the effects of PL on cell migration or invasion. **P<0.01 vs PL-0 µM and ##P<0.01 vs corresponding PL 10 µM control (n=3).
Piperlongumine induces ROS accumulation to exert its anti-cancer effects on HCC cells
We then examined whether ROS accumulation is required for PL’s anti-cancer effects in HCC cells as previous studies have shown that ROS is the major downstream player of PL’s action in many types of cancer cells . The fluorescent intensity of 2’-,7’-Dichlorofluorescin diacetate (DCFH-DA, a specific ROS indicator) in primary hepatocytes (Fig. 3A) or L-02 cells (Supplemental Fig. 3A) was not altered at 3 h of PL treatment (5, 10 and 20 µM). In HepG2 (Fig. 3B), Huh7 (Fig. 3C) and LM3 cells (Supplemental Fig. 3B), however, the fluorescent intensities of DCFH-DA were evidently enhanced at 1 or 3 h of PL treatment. Statistical analyses demonstrated that the intracellular ROS levels were significantly increased in HepG2 and Huh7 cells at 1 h of PL treatment (Fig. 3B and 3C). Pre-incubation of antioxidant N-acetyl-L-cysteine (NAC, 3 mM) completely abolished the effect of PL on ROS induction at 3 h of PL treatment in LM3 (Supplemental Fig. 3B) or HepG2 cells (Supplemental Fig. 3C). Consistently, intracellular glutathione (GSH) levels were significantly reduced in HepG2 or Huh7 cells at 1 h of PL treatment (Fig. 3D). Pre-treatment of NAC (3 mM) reversed PL’s effect on GSH reduction (Fig. 3D). Pre-treatment of NAC or GSH completely reversed PL-induced cell death in Huh7 cells (Fig. 3E) and HepG2 cells (Fig. 3F) while delayed administration of antioxidants partially reversed PL-induced cell death in HCC cells at a time-dependent manner (Fig. 3E and 3F). In addition, administration of NAC completely reversed PL-suppressed cell migration in HepG2 cells after cell scratching (Fig. 3G). Consistently, NAC abolished the PL’s suppression effect on cell migration in the transwell migration assay (left upper panels, Fig. 2D) and cell invasion in the transwell invasion assay (left lower panels, Fig. 2D).
Figure 3: Piperlongumine induced ROS accumulation to kill and suppress HCC cell migration. (A) Effects of PL on the ROS level in primary rat hepatocytes. Cultured primary rat hepatocytes were treated with PL and stained with DCFH-DA. Bar, 20 µm. Representative micrographs showed that the fluorescent intensity of DCFH-DA was not altered within 3 h after PL treatment. For statistical analysis, the mean DCFH-DA fluorescent intensity (representing cellular ROS level) was measured from 9 random fields for each culture. All values represented mean ± SEM of three independent experiments. (B-C) Effects of PL on the ROS level in HepG2 (B) and Huh7 (C) cells. Bar, 20 µm. *P<0.05 and **P<0.01 vs. corresponding PL 0 µM (n=3). (D) Effects of PL on the GSH level in HCC cells. NAC was pretreated for 1 h and then co-treated with PL for another 1 h. *P<0.05, **P<0.01 vs. PL 0 µM control. ##P<0.01 vs. corresponding PL 20 µM control. (E-F) Effects of antioxidants on PL-induced cytotoxicity in Huh7 (E) and HepG2 (F) cells. NAC (3 mM) or GSH (5 mM) was administrated either before PL (20 µM) administration (PL+NAC pre), simultaneously with PL (PL+NAC/GSH) or after PL treatment (PL+ NAC/GSH post). Cell viability was measured by MTT assays. **P<0.01 vs. DMSO control; #P<0.05 or ##P<0.01 vs. PL 20 µM control (n=3). (G) Effects of NAC on PL-suppressed HepG2 cell migration after cell scratching. NAC (3mM) was administrated simultaneously with PL after cell scratching. Bar, 100 µm. **P<0.01 vs. PL 0 µM or PL 10 µM+NAC (n=3).
Piperlongumine induces ER stress-responses which preferentially suppresses HCC cell migration/invasion
The downstream signaling pathways of ROS which is responsible for PL’s anticancer effects remain elusive. We examined the role of ER stress-responses in PL-treated HCC cells as the ROS-ER axis plays a pivotal role in controlling ROS-mediated homeostasis and cellular effects . Western blotting analysis showed that PKR-like ER kinase (PERK, a specific ER-stress responsive protein) was prominently activated in HepG2 (Fig. 4A) and LM3 (Fig. 4B) cells at various time points (e.g., 3 h as indicated by the red rectangular box) of PL treatment in a dosage-depedent manner (0, 5, 10 and 20 µM). Fluorescent cytoimmunostaining assay also clearly showed that the level of phosphorylated PERK (p-PERK) was evidently increased in Huh7 (Fig. 4C) and HepG2 cells (Supplemental Fig. 4A) at various time points (3, 6 and 12 h) of PL treatment (20 µM). In addition, other ER-endocking proteins such as glucose-regulated protein 78 (Grp78, i.e., BiP) and inositol-requiring enzyme-1α (Ire 1α) were also clearly up-regulated at various time points of PL treatment in HepG2 (Fig. 4A) and LM3 (Fig. 4B) cells. Consistently, the downstream product of ER stress-response, CHOP, was also up-regulated evidently at various time points of PL treatment in HepG2 (Fig. 4A and Supplemental Fig. 4A), LM3 (Fig. 4B) and Huh7 cells (Fig. 4C). ER is a major organelle storing Ca2+ which is released into cytosol to activate downstream signaling pathways upon various stimuli including ROS . In primary rat hepatocytes, the concentration of cytoplasmic free Ca2+ as measured by Fluro-3 AM (a specific fluorescent probe for free calcium) was not altered at 3 h of PL treatment (0, 5, 10 and 20 µM) (upper panels, Fig. 4D). In HepG2 (middle panels, Fig. 4D) and Huh7 (lower panels, Fig. 4D) cells, however, the concentration of cytoplasmic free Ca2+ was prominently increased at 3 h of PL treatment in a dose-dependent manner (0-20 µM). These evidences together verified that PL induced ER stress-responses in HCC cells.
We then examined the roles of ER stress-responses in PL’s anticancer effects in HCC cells by using specific ER stress-response inhibitor 4-phenylbutyric acid (4-PBA) [29, 30]. MTT assay revealed that 4-PBA did not alter PL-induced cell viability reduction in HepG2 or LM3 cells (Supplemental Fig. 5A and 5C). Consistently, results of PI staining showed that 4-PBA did not alter PL-induced cell death in HepG2 cells (Supplemental Fig. 5B). However, 4-PBA completely abolished PL-suppressed cell migration in HepG2 (Fig. 4E) and Huh7 cells (Supplemental Fig. 6) at various time points after scratching. Consistently, 4-PBA significantly reversed PL-suppressed HepG2 cell migration in transwell migration assays and HepG2 cell invasion in the transwell invasion assays (Fig. 2D). The reversing effects of 4-PBA on PL’s effects on HCC cell migration were close to those of NAC, suggesting that PL inhibited HCC cell migration or invasion via ROS-ER-mediated signaling preferentially.
Figure 4: Piperlongumine induced ER stress-responses to suppress HCC cell migration preferentially. (A-B) Representative results of Western blotting analysis of specific ER stress-response proteins in PL-treated HepG2 (A) and LM3 (B) cells. HepG2 or LM3 cells were treated with 0, 5, 10 and 20 µM of PL for 3, 6 or 12 h. Equal amounts of total proteins were subjected to Western blotting analysis with designated antibodies. The red box indicated the most evident changes of p-PERK, Grp78, Ire 1α or CHOP in PL-treated HepG2 (A) and LM3 (B) cells. (C) Representative micrographs of fluorescent immunostaining showed PL (20 µM) activated p-PERK and up-regulated CHOP in Huh7 cells. Bar, 20 µm. (D) Representative fluorescent micrographs of Fluro-3AM showed that PL enhanced free calcium concentration in PL-treated HepG2 or Huh7 cells but not primary rat hepatocytes. Bar, 20 µm. (E) Effects of ER stress-response inhibitor 4-PBA on PL-suppressed HepG2 cell migration after cell scratching. Bar, 100 µm. **P<0.01 (n=3).
Piperlongumine activates MAPKs signaling pathways which preferentially suppress HCC migration
MAPKs are canonical ROS- and ER-stress responsive signaling pathways that control cell death under oxidative stress [31, 32]. Results of Western blotting analysis showed prominent activation of p38, JNK and Erk in HepG2 (Fig. 4A) and LM3 (Fig. 4B) cells at various time points of PL treatment in a dose-dependent manner (0, 5, 10 or 20 µM). We then examined whether the activation of MAPKs contributed to PL’s anti-cancer effects in HCC cells. Pharmacological inhibition of p38, JNK or Erk signaling pathway by using corresponding inhibitor SB203580, SP600125 or U0126 did not alter PL’s effects on cell death in HepG2 (Fig. 5C) or LM3 cells (Fig. 5D). However, suppressing p38 (SB203580), JNK (SP600125) or Erk (U0126) signaling pathway prominently reversed PL’s effects on HepG2 cell migration at 24 or 48 h after cell scratching (left panels, Fig. 5E). Statistical analysis demonstrated that the wound sizes were significantly increased in SB203580/ SP600125/U0126 plus PL 10 µM treatment groups as compared to that in PL 10 µM treatment group in HepG2 cells (right panel, Fig. 5E). These evidences suggested that the activation of MAPKs mainly contributed to PL’s effect on cell migration.
Figure 5: Piperlongumine activated MAPKs to suppress HCC cell migration preferentially. (A-B) Representative results of Western blotting analysis demonstrated that p38, JNK and Erk were prominently activated in PL-treated HepG2 (A) and LM3 (B) cells in a dosage-dependent manner. (C-D) Effects of specific inhibitors on the cell viability in PL-treated HepG2 (C) and LM3 (D) cells as measured by MTT assays. PL were administrated alone or together with other drugs (i.e., SB203580 10 µM, SP600125 10 µM, PD98059 10 µM, LY294002 10 µM, U0126 10 µM, Go6983 10 µM or 3-MA 10 µM). *P<0.05, **P<0.01 vs. PL 0; #P<0.05, ##P<0.01 vs. corresponding PL 20 (n=3). (E) Effects of specific MAPKs inhibitors on PL-suppressed HepG2 cell migration after cell scratching. PL (10 µM) was administrated together with 10 µM of SB203580, SP600125 or U0126. Bar, 100 µm. **P<0.01 (n=3).
Piperlongumine functions through ROS-ER-MAPKs-CHOP axis in HCC cells
We then verified the existence of ROS-ER-MAPKs-CHOP axis in PL-treated HCC cells by using the corresponding inhibitors. Results of Western blotting analysis showed that co-incubation of antioxidant NAC with PL completely abolished PL-activated PERK (Fig. 6A, indicated by the rectangular box) and p38/JNK/Erk (Fig. 6B), and PL-up-regulated Ire 1α and CHOP (Fig. 6A) in HepG2 cells at various time points of PL treatment. The ER stress inhibitor 4-PBA showed similar inhibitory effects on ER-MAPKs-CHOP signaling pathways compared to NAC (Fig. 6A and 6B). Consistently, results of fluorescent cytoimmunostaining assay also showed that NAC and 4-PBA evidently reduced the elevated level of p-PERK (Supplemental Fig. 4B) or CHOP by PL (Fig. 6C) at various time points of PL treatment in HepG2 cells. Further, suppressing p38/JNK/Erk signaling pathways by SB203580/SP600125/U0126 correspondingly evidently reduced PL-elevated CHOP at 6 or 12 h of PL treatment in HepG2 cells (Fig. 6C). These evidences demonstrated that PL activated ER-MAPKs-CHOP axis signaling pathways via ROS-dependent mechanisms.
Finally, we examined the role of CHOP in HCC cell migration by knocking-down CHOP via small RNA interference technique. Transfection of small interfering RNA specific to CHOP (si-CHOP)  effectively reduced the expression of endogenous CHOP in HepG2 cells (Supplemental Fig. 7A) but did not affect the viability of HepG2 cells (MTT assay) at 24 h of PL treatment (Supplemental Fig. 7B). However, transfection of si-CHOP evidently accelerated HepG2 cell migration at various time points (12, 24 and 36 h) after cell scratching as compared to corresponding si-GFP (green fluorescent protein) controls (Fig. 6D). Statistical analysis demonstrated that knocking-down of CHOP (si-CHOP) significantly reduced the wound-size of HepG2 culture at various time points after scratching without or with PL treatment (PL 0, Fig. 6E and PL 10 µM, Fig. 6F) as compared to corresponding si-GFP controls. These results supported that CHOP was required for suppressing HCC cell migration and was a critical downstream target of PL’s action in HCC cell migration.
Figure 6: Piperlongumine suppressed HCC cell migration via ROS-ER-MAPKs-CHOP signaling pathway. (A) Representative results of Western blotting analysis showed the effects of NAC or 4-PBA on the expression of specific ER stress-response proteins (p-PERK/PERK, Ire 1α and CHOP) in PL-treated HepG2 cells. The red boxes indicated the most evident reversal effect of NAC or 4-PBA. (B) Representative results of Western blotting analysis showed the effects of NAC or 4-PBA on the activation of p38/JNK/Erk. (C) Representative micrographs of fluorescent cytoimmunostaining showed the effects of NAC, 4-PBA or MAPKS inhibitors on CHOP expression in PL-treated HepG2 cells. Bar, 20 µm. (D) Effects of knocking-down CHOP by RNA interference on HepG2 cell migration. HepG2 cells were transfected with small interfering RNA specific for CHOP (si-CHOP) for 2 days and then subjected to cell scratching and PL treatment. Small interfering RNA specific for green fluorescent protein (si-GFP) was used as non-specific control. Bar, 100 µm. (E) and (F) Statistical analyses demonstrated that silencing CHOP (si-CHOP) significantly accelerated the speed of wound-healing of HepG2 cells after scratching without (E) or with PL treatment (F). *P<0.05, **P<0.01 vs. corresponding si-GFP (n=3).
Piperlongumine activates ER-MAPKs-CHOP axis signaling and reduces HCC in vivo
After clarifying the anticancer effects and mechanisms of PL in HCC cells in vitro, we examined the role of PL in HCC-bearing mice. Equal amount of murine ascetic H22 hepatoma cells was subcutaneously inoculated into the flank side of adult male Kunming mice [33-35]. After 24 h of inoculation, PL was administrated once a day by intraperitoneal (i.p.) injection for 14 days consecutively and then tumor xenografts were isolated. (Fig. 7A) showed that the tumor xenografts from PL-treated groups were evidently smaller than those from DMSO control group. Statistical analysis demonstrated that the average volume (Fig. 7B) and weight (Fig. 7C) of the tumor xenografts from PL treatment groups were both significantly decreased as compared to that from DMSO control group. Notably, PL at a much lower concentration (1.5 mg/kg) showed a comparable anticancer effect in HCC-bearing mice and increasing PL concentration did not significantly enhance its anticancer effects (Fig. 7A, 7B and 7C).
Consistent with in vitro assays, results of Western blotting analysis demonstrated that PL significantly activated p-PERK and up-regulated Ire 1α, PDI, and CHOP in H22 tumor xenografts (Fig. 7D). Meanwhile, MAPKs (i.e., p38, JNK and Erk) were significantly activated in H22 tumor xenografts (Fig. 7E). These results verified that the ER-MAPKs-CHOP signaling pathway was activated by PL in HCC cells in vivo.
Figure 7: Piperlongumine suppressed HCC development and activated ER-MAPKs-CHOP signaling pathway. (A) Representative inoculated HCC tumors from mice treated with PL. Equal amounts of H22 cells were subcutaneously injected into the left flank of adult mice. PL, 5-Fu or DMSO was administrated for 14 days (once per day) and then the tumor xenografts were removed completely from tissues. (B-C) Statistical analyses demonstrated that the average volume (B) and weight (C) of HCC xenografts from PL-treated mice were significantly reduced. *P<0.05, **P<0.01 vs. DMSO group (n=8). (D) Representative Western blotting analysis and statistical analysis of p-PERK/PERK, Ire 1α, PDI and CHOP from xenografts. *P<0.05, **P<0.01 vs. corresponding DMSO control (n=5). (E) Representative Western blotting analysis and statistical analysis of p-p38/p38, p-JNK/JNK and p-Erk/Erk from xenografts. *P<0.05, **P<0.01 vs. corresponding DMSO control (n=5).
In the present study, we demonstrated that PL selectively killed HCC cells and inhibited their migration and invasion. PL activated ROS-ER-MAPKs-CHOP signaling axis prominently, which consequently suppressed HCC cell migration/invasion preferentially.
We demonstrated that PL was a selective anti-cancer drug for HCC. PL effectively induced cell death in various HCC cell lines including the highly malignant LM3 cells  with an IC50 of 10-20 µM but had little cytotoxicity in normal hepatic cells under similar conditions (Fig. 1). The cytotoxic efficacy of PL in HCC cells was similar to that in glioblastoma multiforme (GBM) cells , suggesting a general selective cytotoxicity of PL in those highly malignant cancers. In addition, PL selectively suppressed HCC migration/invasion but did not affect the migration of normal hepatic cells (Fig. 2). Notably, PL at a lower concentration (2.5-10 µM) exhibited a remarkable effect on suppressing HCC cell migration in scratched cultures in which PL-induced cell death was negligible (Fig. 2), suggesting a major anti-cancer effect of PL on suppressing metastasis. This is particularly important for the treatment of highly malignant tumors such as HCC since metastasis is the major cause of death [1-3]. Consistent with the anti-cancer effects of PL in vitro assays, PL was effective in reducing HCC development in mice (Fig. 7). PL might suppress HCC development in vivo via inducing cell death, suppressing cell migration/invasion and reducing tumor vessel formation (Supplemental Fig. 8). PL at 1.5-3.5 mg/kg/d showed comparable anticancer effects in HCC xenograft-bearing mice, suggesting that the therapeutic effects of PL in vivo reached a plateau at the dosage around 1.5 mg/kg/d and that further increasing the dosage of PL might not improve PL’s therapeutic effect much due to the limited effective drug concentration in cancer tissues, the varied sensitivity of cancer cells to drug, the complicated feedback drug-resistant mechanisms or the increased side effects of drug in vivo. Since PL is a natural product which has been already used in traditional medicines for a thousand years [12, 13], it is practical to apply PL to the treatment of the highly malignant HCC clinically.
Our data clearly demonstrated that the ROS accumulation was a major initial factor responsible for PL’s anticancer effects in HCC as pre-treatment of antioxidants completely abolished PL-induced cytotoxicity in HCC cells while delayed administration of antioxidants (3-12 h post PL treatment) markedly reduced their efficacy in antagonizing PL’s cytotoxicity (Fig. 3E). Previous studies mainly focus on the cytotoxic effects of PL in cancer cells and various ROS-dependent cell death mechanisms, e.g., apoptosis [36-38], autophagic cell death [16, 39], cell cycle arrest [17, 38], GSTP1 , p38 [9, 39], JNK [9, 38], Erk , which have been proposed in different cancer cell lines. Until now, however, the exact molecular mechanisms of PL’s action in cancer cells remain largely unclear, probably reflecting the nature of non-specific ROS stimulation in multiple signaling pathways and the complicated regulatory mechanisms involving in cell death process [5-9]. In HCC cells, the PL-induced cytotoxicity could not be efficiently reduced by a single signaling pathway inhibitor except antioxidants, suggesting that multiple ROS-dependent cell death mechanisms are involved in PL’s cytotoxicity in HCC cells.
Unlike the elusive cell-death mechanisms underlying PL’s action, we clarified a key signaling pathway (i.e., ROS-ER-MAPKs-CHOP) which was preferentially responsible for PL’s action on HCC cell migration/invasion. Different from previous studies which showed a differentially activation of p38, JNK and Erk in distinct cancer cells [9, 10, 15], we demonstrated that PL induced prominent activation of all the three major MAPKs signaling pathways including p38, JNK and Erk in HCC cells, supporting that MAPKs were major ROS-stress responsive signaling pathways upon PL treatment. It is well-known that ER is a major organelle responsible for ROS accumulation  and MAPKs are canonical downstream signaling pathways of ER stress-responses . Indeed, ROS-dependent activation of PERK, release of Ca2+ and induction of Ire 1α were prominent in HCC cells upon PL treatment (Fig. 4). Inhibition of ER stress-responses by 4-PBA suppressed PL-activated MAPKs (Fig. 6), supporting that PL activated the MAPKs via ER stress-responses in HCC cells. CHOP is a key ER stress-response product involved in cell death . Interestingly, suppressing ER-MAPKs-CHOP signaling axis by either inhibitors (4-PBA, SP, SB, or U0126) or knocking-down of CHOP did not affect the rate of HCC cell death evidently but showed prominent effects on HCC cell migration, suggesting that PL preferentially suppressed HCC migration/invasion via ER-MAPKs-CHOP. Consistent with PL’s effects on HCC cell migration, PL also suppresses cell migration of highly malignant GBM cells via ROS-p38/JNK/NFκB signaling . These evidences suggest that MAPKs might be a signaling hub for PL’s suppressed effects on cell migration/invasion. Further dissecting the molecular mechanisms underlying PL’s effects on cancer cell migration/invasion is important for future applications of PL in cancer therapy.
In summary, we have demonstrated that PL selectively killed HCC cells and suppressed HCC cell migration/invasion. Preferentially, PL suppressed HCC migration/invasion via ROS-ER-MAPKs-CHOP axis signaling. Our data suggest that PL has the prospects of application in the treatment of highly malignant HCC.
Cell culture, reagents and antibodies
Human HCC cell lines (HepG2, Huh7) and normal hepatic L-02 cells were purchased from ATCC (Manassas, VA, USA). HepG2 and Huh7 were cultured with 10% FBS in DMEM and L-02 was cultured with 10% FBS in RPMI-1640. Highly malignant human HCC LM3 cells were obtained from Zhongshan Hospital, Fudan University and cultured with 15% FBS in DMEM . Mouse HCC cell line H22 was purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Primary cultures of rat hepatocytes were set as reported . Briefly, hepatocytes (cell viability >85%) were isolated from livers of normal male Wistar rats by collagenase perfusion and mechanical disruption, seeded in collagen-coated 96-well plates with 10% FBS in high-glucose DMEM at a density of 2×104 cells/cm2, and used at day 5 in vitro. All culture media and fetal bovine serum (FBS) were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). Piperlongumine, 5-fluorouracil (5-FU), 4-phenylbutyric acid, Hoechst 33342, propidium iodide, Fluo-3 AM, trypan blue, glutathione, N-acetyl-L-cysteine (NAC), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-Methyladenine (3-MA) and crystal violet were purchased from Sigma-Aldrich (St Louis, MO, USA). SB203580 (p38 pathway inhibitor), SP600125 (JNK pathway inhibitor), U0126 or PD98059 (MEK 1/2 inhibitors), LY294002 (PI-3K inhibitor) and Go6983 (PKCζ inhibitor) were purchased from Cell Signaling Technology (Danvers, MA, USA). Annexin V-FITC apoptosis detection kit was purchased from KeyGEN BioTECH (Nanjing, China). Quantification of reduced GSH was conducted by a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against Grp78, PDI, p-PERK (Thr981)/PERK, Ire 1α, CHOP, p-p38 (Thr180/Tyr182)/p38, p-JNK (Thr183/Tyr185)/JNK, p-Erk (Thr202/Tyr204)/Erk were from Cell Signaling Technology (Danvers, MA, USA).
Piperlongumine and other drug treatments
PL was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 50 mM and used at designated final concentration (0-20 µM) with a maximal DMSO concentration less than 0.5% DMSO. DMSO of the same concentration in DMEM was used as the vehicle control (i.e., PL 0). NAC, GSH and SB203580/SP600125/U0126 were used at 3 mM, 5 mM and 10 µM respectively. 4-PBA was used at a final concentration ranged from 1-5 mM. NAC and other drugs were added simultaneously with PL at designated final concentrations or as indicated.
Cell viability or cell death assays
Cell viability was determined by MTT assay and cell death was determined by trypan blue exclusion assay or PI staining. For MTT assays, 5000 cells/well were seeded in 96-well plates. After 24 h of incubation, cells were treated with drugs in six parallel wells for designated times and then assayed with MTT as reported [42, 43]. For trypan blue exclusion assay and PI staining, cells at 60–70% confluence in six-well plates were treated with drugs in triplicates for 24 h and then subjected for corresponding assays.
Flow cytometry assay
FCM assay was employed to analyze apoptosis and cell cycle as previously reported . HCC cells were treated with PL for 24 h and co-stained with annexin V-FITC and PI according to the manufacturer’s instructions (KeyGEN BioTECH). Apoptotic cells were separated and quantified by a FACSCalibur Flow Cytometry System (Becton Dickinson, San Jose, CA, USA). For cell cycle assay, HCC cells were treated with PL for 24 h and stained with PI .
Scratch-wound healing assay
Cell migration after scratching was performed as reported previously . Briefly, confluent cells in 35-mm dishes were scratched with 200-µl pipette tips. Immediately after cell scratching, the wounded edges were micrographed (designated as 0 h). Scratched cells were then incubated with fresh complete media with appropriate concentration of drugs and the wounded edges were micrographed at designated time points. The wound size (i.e., the gap between the two opposite wounded edges) was calculated using the Image-Pro Plus software (Media Cybernetics, USA) and the average of wound size represented at least 12 different fields in each culture dish. Relative wound sizes of designed time points after scratching were compared to that of 0 h control.
Transwell migration and invasion assays
Transwell migration and invasion assays were performed as reported [10, 44]. Briefly, HepG2 cells (1×105 cells/well) were seeded into the upper chamber of a transwell apparatus with a 8-µm pore size membrane (Corning Incorporated, Corning, NY, USA) in 200 µl of serum-free DMEM containing drugs. For the invasion assay, the upper chamber was pre-coated with matrigel basement membrane matrix (BD Bioscience, Bedford, MA, USA) according to the manufacturer’s protocols. For both assays, the medium of lower chamber contained 800 µl of complete medium. After 24 h, the migrated or invaded cells at the lower surface of the filter were stained, micrographed and calculated as reported previously . The average migrated/invaded cell number represented at least nine different fields in each filter.
Measurement of intracellular ROS and reduced glutathione (GSH)
Intracellular ROS level was measured using DCFH-DA as reported previously [9, 10]. Briefly, PL-treated cells were incubated with 10 µM of DCFH-DA for 30 min at 37°C, washed twice with PBS and then micrographed with a conventional fluorescent microscope (Olympus, Tokyo, Japan). For each culture, a minimum of 9 random fields were captured. Average fluorescent intensity was analyzed using the Image-Pro Plus software (Media Cybernetics, USA). For GSH measurement, PL-treated HepG2 or Huh7 cells were washed with ice-cold PBS three times, scrapped off from the plates and subjected to sonification. The supernatants were collected and reduced GSH was calculated according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute).
Western blotting analysis and fluorescent cytoimmunostaining
Western blot analysis and fluorescent cytoimmunostaining were performed as described previously . Briefly, equal amounts of total proteins were subjected to mini-PAGE gel electrophoresis and transferred to NC membranes. The blots were incubated with corresponding primary and fluorescent secondary antibodies. The bands were quantified using the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA). β-actin was used as internal control. For fluorescent cytoimmunostaining, PL-treated cells in the 35-mm culture dishes were fixed, permeabilized, blocked, and then incubated with corresponding primary and Dylight 488-labeled secondary antibodies (Abbkine, Redlands, CA, USA). Micrographs were taken under the same conditions with a conventional fluorescent microscope (Olympus, Tokyo, Japan).
Intracellular Ca2+ measurements
Intracellular Ca2+ levels were detected with Fluo-3 AM as reported . PL-treated cells were washed with Ca2+-free PBS three times and then incubated with 5 μM of Fluo-3AM for 30 min. After brief rinse with Ca2+-free PBS, living cells were micrographed immediately under a conventional fluorescent microscope (Olympus, Tokyo, Japan).
Small interfering RNA for CHOP (5’-GGUAUGAGGACCUGCAAGA-3’, si-CHOP) was chemically synthesized (Guangzhou RiboBio Co., Ltd., Guangzhou, China) and non-specific control si-GFP was provided by Guangzhou RiboBio. Twenty-four hours after cell seeding, interfering RNAs at 50 nM were transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as previously reported . Two days after siRNA trensfection, the cultures were subjected to PL treatment and the corresponding assays.
Hepatocarcinoma 22 (H22)-derived solid HCC in mice
Specific pathogen-free male Kunming mice with body weights of 18-22g were purchased from the Experimental Animal Centre, Huazhong University of Science and Technology (HUST). All the mice were housed in a clean environment and had free access to standard rodent diet and water at room temperature and a humidity of 45–55%. All the animal experimental procedures are approved by the Animal Care and Use Committee of Tongji Medical College, HUST. H22-derived solid HCC in mice was generated by injecting 2×107 H22 cells (in 0.2 ml of PBS) into the left armpits of mice as described . Twenty-four hours after initial H22 inoculation, 50 mice were randomly divided into five groups (i.e., DMSO, PL 1.5 mg/kg.day, PL 2.5 mg/kg.day, PL 3.5 mg/kg.day and 5-FU 20 mg/kg.day; 10 mice/group) and PL or 5-Fu was delivered as designated by i.p. once per day for 14 days consecutively. H22-derived tumor xenografts were harvested, measured and weighted on the 15th day of H22 inoculation as described previously .
All experiments were repeated independently at least three times. The values were expressed as mean±SEM and statistics were performed with a 2-way ANOVA followed by the Student-Newman-Keuls test. P values of less than 0.05 were considered statistically significant.
This work was supported by grants from the National Nature Science Foundation of China (Grant No. 81172397, 81471386) and the Fundamental Research Funds for the Central Universities, HUST (No. 2013ZHYX017) to X.Q. Chen and the China Postdoctoral Science Foundation (No. 2013M542026) to F Pan.
Conflict of interest
4-PBA, 4-phenylbutyric acid; 5-FU, 5-fluorouracil; CHOP, C/EBP homologous protein; DCFH-DA, 2’-,7’-Dichlorofluorescin diacetate; 3-MA, 3-Methlylaclenine; DMEM, Dulbecco’s modiﬁed Eagle’s medium; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; Erk, extracellular signal-regulated kinase; FBS, fetal bovine serum; FCM, flow cytometry; GFP, green fluorescent protein; Grp78, glucose-regulated protein 78; GSH, glutathione; HCC, hepatocellular carcinoma; JNK, c-Jun N-terminal kinase; IC50, half maximal (50%) inhibitory concentration; i.p., intraperitoneal; Ire 1α, inositol-requiring enzyme 1 alpha; MAPKs, mitogen-activated protein kinases; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-L-cysteine; NFκB, nuclear factor kappa B; PBS, phosphate buffered saline; PDI, protein disulfide isomerase; PERK, PKR-like ER kinase; PI, propidium iodide; PL, piperlongumine; ROS, reactive oxygen species; siRNA, small interfering RNA.
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domain: medical


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Separate names with a comma.
Discussion in 'Introductions' started by DFlynt, Oct 1, 2013.
Hi all, here to learn about the uses of paracord.
Greetings and welcome to the forum!
I like your avatar!
Welcome to the forum!
Welcome DFlynt! A question for you...do you have any relatives in Tennessee?
Wweellccoommee,can you crack the code?
Sorry no I don't.

domain: geography


text:
Day 13 - Watercolour
This is my second attempt at a watercolour mani and while it wouldn't be my design of choice it worked out better than the last time.
Sinful Colors - Snow Me White
Sinful Colors - Mint Apple
Bondi - City Slicker
Barry M - Papaya
Be sure to check out these lovely ladies...
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Kate FishingBeauty - http://fishing4beauty.blogspot.com/
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Me (Ameerah Muhammad) - http://www.valiantlyvarnished.com/
Gillian Ni Fhaolain - http://email@example.com

domain: artistic


First comes China, then ... Japan?
While smaller than it's giant neighbor to the West, Japan is becoming the next frontier in the increasingly global fight between Uber, Lyft, Didi Kuaidi and others to dominate the fast-growing ride-hailing business.
While Japan's market is much smaller than India's or China's — 127.3 million potential consumers versus the 1.3 billion in China — the need for transportation alternatives and demographic realities presents a big opportunity.
Today, residents of Tokyo have buses, subways and taxis as transportation options. The subway lines are competing, privately owned companies often forcing commuters to switch lines halfway through their ride.
Taxis can be prohibitively expensive, leading many potential riders to rent hotel capsules for the night rather than travel across town. This leaves a gap between limited but affordable public transportation, and costly private car or taxi transport.
Enter e-hailed car rides.


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Many factors are used in deciding whether to close school and how to make up missed days. Atkins Superintendent Mark Gotcher said one factor in deciding whether or not to close is more important than the rest.
"It's always about student safety," he said.
When inclement weather is forecasted, the superintendents in Pope County keep in contact with each other and, when possible, decide together how to proceed. Superintendents also seek input from school board members.
"If all the experts say it's definitely going to happen, the team makes the decision to close," Gotcher said.
Once the decision to close has been made, it takes Gotcher about 30 minutes to contact everyone.
"The first step is school messenger and Facebook," he said. "School messenger alerts staff and parents. Then I contact the media."
Gotcher said the hardest decision to make is closing early with the kids at school.
For one thing, the district must ensure buses can run early before it can announce an early dismissal.
Gotcher said the transportation director tells bus drivers to be on standby when bad weather could cause schools to release early. Some drivers have full-time jobs they aren't able to leave, so the district has several emergency substitute drivers.
"It's a difficult decision," Gotcher said. "If you wait, will it get worse or better? Usually it's going to get worse."
If the school closes before the students receive six hours of instruction, the day does not count. Districts budget their calendars for five snow days each year, but 2014 has been an unusually active season causing many districts to re-organize.
Because staff contracts are tied to the district calendar, any changes have to be voted on by a majority of staff members and approved by the school board.
The Arkansas Department of Education (ADE) offers the following options for making up missed days:
• Use the five inclement weather days built into the calendar.
• Use previously scheduled teacher work days as instructional days.
• Hold instructional days on upcoming scheduled holidays.
• Use part or all of spring break.
• Add days to the end of the school year.
Districts are allowed to submit other proposed options to the ADE for approval.

domain: news


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Babatunde Fowler. NNPC: Oil price in Nigeria cheapest in West Africa. The group managing director (gmd) of The Federal inland revenue service (firs) says its Executive Chairman, Babatunde Fowler, has been invited to participate in the global conference on how to curtail Illicit Financial Flows.
Another Snowbird participant, Ken Schwaber-the cofounder of Scrum and founder of Scrum.org-says waterfall “literally ruined. first evening of the retreat, telling her, “There’s a powerful odor of.
Group of FIRS Staff detail how Fowler allegedly ruined FIRS.
Babatunde Fowler, Executive Chairman, Federal Inland Revenue Service (FIRS) has reiterated call on eligible citizens to ensure adequate payment of tax to government for enhanced revenue generation.
Dr Babatunde Fowler (born 12 August 1956) is a Nigerian public officer, tax administrator and social reformer. He was the Executive Chairman Lagos State Board of Internal Revenue and Chief Executive Officer Lagos State Internal Revenue Service. He is presently the Executive Chairman of the Federal Inland Revenue Service (FIRS).
The FIRS chairman Babatunde Fowler is thought to have been nominated by APC chieftain Bola Tinubu to try and recreate the Lagos miracle at the center. However, going by complains from disgruntled staff, it seems the employees of the agency may not totally be on board with him making it.
Concerned Staff Detail How Fowler Allegedly Ruined FIRS, Ask Buhari Not To Re-appoint Him June 9, 2019 June 9, 2019 newsadmin A group, Concerned Staff of FIRS, have petitioned President Muhammadu Buhari over the atrocities and infractions allegedly perpetrated by the Executive Chairman of the Federal Inland Revenue Service, Babatunde Fowler.
How Babatunde Fowler ruined FIRS – Group June 11, 2019 – by Parrot Nigeria – Leave a Comment A group, Concerned Staff of FIRS, have petitioned President Muhammadu Buhari over the atrocities and infractions allegedly perpetrated by the Executive Chairman of the Federal Inland Revenue Service, Babatunde Fowler.
How to Use Dave Ramsey’s Budget Percentages Two major banks slashed interest rates on your savings accounts It remains to be seen how much of the latest cut will be passed through to mortgage rates. anz was the first major bank to react. always factor in a two-to-three percentage point buffer, on top of.In this post I have provided some recommended budget percentages so as to simplify your budgeting experience. Another thing that will help simplify budgeting using of one of the best pieces of budgeting software. If you need more budgeting help you might want to check out The Secret to a Successful Budget.
Babatunde Fowler, Executive Chairman of the Federal Inland Revenue Service A group, Concerned Staff of FIRS, have petitioned President Muhammadu Buhari over the.
He had a 7-under 63, making him the first player in 101 years of the PGA. a double bogey on the 10th hole for a 69 and was in a group that included Dustin Johnson, Phil Mickelson, Rickie Fowler and.
China’s home prices growth fastest in five months, raises policy challenge – ET RealEstate 2 Myths Holding Back Home Buyers · Freddie Mac recently released a report entitled, “Perceptions of Down Payment Consumer Research.” Their research revealed that, “For many prospective homebuyers, saving for a down payment is the largest barrier to achieving the goal of homeownership. Part of the challenge for those planning to purchase a home is their perception of how much they will need to save for the.Last night, China published May, 2017 new home pricing data for its 70 city index, which showed a continued slowing in growth: a key driver for commodity consumption inside China. China’s New home price growth slows Further in May: Should You Be Concerned | Zero Hedge
How to successfully import data into Sage 50 U.S. Edition Managing records and transaction postings into Sage 50 U.S. Edition

domain: economics


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Mulata (C76) [Dokuritsu Gurentai (Bow Rei)] Chinami Series [Tokubetsu Hen] Mama wa Shougaku Rokunensei Analsex
(C76) [独立愚連隊 (某零)] 千波シリーズ【特別編】ママは小○六年生
54 pages - Uploaded
#145648 - Sam grinned and nodded. ” I looked at the clock in the bathroom. ” I hurried and pulled his jeans down his legs, then from around his feet.

domain: literature


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Stay Sexy & Don’t Get Murdered Pdf sharing never before heard stories ranging from their struggles with struggles. It has the story of the failures and struggles of a girl. Who is trying to focus that how she will take control of these situations. You can download Stay Sexy & Don’t Get Murdered Pdf from our website for a free read.
Stay Sexy & Don’t Get Murdered Pdf Review:
Sharing never-before-heard stories ranging from their struggles with depression, eating disorders, and addiction, Karen and Georgia irreverently recount their biggest mistakes and deepest fears. Reflecting on the formative life events that shaped them into two of the most followed voices in the nation.
In Stay Sexy & Don’t Get Murdered, Karen and Georgia focus on the importance of self-advocating and valuing personal safety over being ‘nice’ or ‘helpful.’ They delve into their own pasts, true crime stories, and beyond to discuss meaningful cultural and societal issues with fierce empathy and unapologetic frankness.
About The Author:
Georgia Hardstark: Georgia Hardstark is an American television host and podcast personality. She is the co-host of the true-crime comedy podcast My Favorite Murder along with comedian Karen Kilgariff. In 2018, Hardstark and Kilgariff co-founded the Exactly Right podcast network.
Stay Sexy & Don’t Get Murdered Pdf Features:
- We are bringing Stay Sexy & Don’t Get Murdered Pdf on our website with high-quality fonts
- This book has 304 pages and got an excellent user score of 4.10 on Goodreads

domain: library


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On this occasion we didn’t go into the town but just did a walk. There is a really nice one, via the Le Gouffre Cafe and then to the cliffs.
The airport – EuroGA database report – is nice and friendly as always, although the “handling” (in reality there was none, as is normal) has gone up from £10 to £30 over 3 years. Landing fee was £22. Still ok for the occassional trip but at this rate their piston GA traffic will soon disappear.
Two GAR forms on 12hr PN, plus one GAR for Guernsey which goes to two addresses on their website and the ASG address as well. The days when one could go at short notice via Cherbourg are gone, since Cherbourg lost the battle with French police and their bizzare work-minimisation politics and is now 24hr PN and 48hr for weekend trips…
I filed EGKA GWC KATHY ORTAC EGJB 5000ft which is OCAS, so knew that London Control won’t touch it, and neither will Solent who have stopped providing a service to non-CAS-transit traffic. So one flies “VFR 7000”, in VMC or IMC as usual in the UK system, and speaks to Bournemouth 119.480, and then to Jersey Zone who do it correctly, with a squawk etc. At Guernsey I got a vectored ILS 09.
Parked at ASG; no other option AFAIK
ASG has a lot of Rockwell Commanders there, and of course a lot of 2-reg aircraft generally. I think a lot of Commander owners fly to Guernsey for maintenance.
The walk to the cafe
Crab sandwiches; unfortunately only white bread available
The cliff walk
This is a well known beach with another cafe but we didn’t go there because it was a long way down and then a long way back up, and time was a bit limited
The walk back towards the airport
A very English pub, with “all day breakfast” (probably 30000 calories and fried in W80) and tables which your hands get stuck to
This is probably Hurn
and this is definitely Alderney, in a lot of haze which took a bit of work in Lightroom
A nice day trip and a walk, and much nicer than a taxi into town
Alderney is very very nice. Like England in the 1920s Do a search here for EGJA, in our database and I have one here.
Guernsey is different, and Jersey is different again.
I think Guernsey would be great for a EuroGA meet-up and with a cliff walk like above, not a taxi trip into town and a big lunch, which is nothing really special. Some of us used to do these but it seems most Brit long term flyers packed up and disappeared with the virus. And very few people from the mainland fly to the Channel Islands (no idea why this is).
I have Guernsey in the diary for the first weekend in July. Taking some friends so four-up in the TB10 and three hours fuel = MTOW.
All three of the islands make for a good trip. At Guernsey you use ASG, a ‘bizjet handler’ but the fee (although irritating if a for a burger run) is not significant in the context of a weekend away for four. Jersey is via the Aeroclub, so doesn’t cost much. Alderney is just the airport itself I believe. I’ve been to each of the islands a few times and none has ever costed enough to worry about.
is not significant in the context of a weekend away for four
Indeed, but when I posted a trip report in the various GA chat parts of FB (which I do to promote EuroGA ) it drew the usual “what????” response from UK pilots
Nice idea Peter.
I have flown to Alderney and Guernsey a few times now from EGSX.
Great trips out.
I’m up for a meet up!
I’m still amazed that people moan about £30 for landing and handling fees etc when they have all the services of an international airport to hand and they have just burnt £££ to get there
via the Le Gouffre Cafe and then to the cliffs.
Under new ownership for a few months so back to being as good as it used to be
Fuel prices for May 2023 at ASG
Avgas is back in Guernsey !!!
Unfortunately still none at Alderney.


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Black transgender woman, Brianna Hill, shot to death in Kansas City
Yet another Black transgender woman has been murdered this year
This week a Black transgender woman was found shot to death in Kansas City, making her the 21st nationwide this year.
According to local station KCTV5 News, Monday, Brianna “BB” Hill, was allegedly murdered by an unidentified man who currently in police now in custody.
Hill, who goes by the nickname Be’Be was pronounced dead when police arrived at the scene shortly after the shooting. Kansas City police Capt. Tim Hernandez says the alleged shooter was still at the scene when officers arrived. As a result, police are not looking for any more suspects.
“That’s part of my family because we are out here, we are trying to survive and people take action against us, nationwide,” said George Cherry, a neighbor of Hill’s, who says her death hit close to home for him as a gay man.
“They are human beings. Quit the violence,” he continued. “This shouldn’t be happening to the LGBTQ community. It hurts our families and our community,”
Brianna “BB” Hill is at least the 21st known trans or gender non-conforming person to be killed this year.
These victims are not numbers – they were people with hopes & plans, dreams for the future, loved ones & communities who will miss them every day. https://t.co/Y41tWPDD7c
— Human Rights Campaign (@HRC) October 20, 2019
The Human Rights Campaign reports Hill is the 21st known transgender or gender non-conforming person killed this year in the United States, and of that 21 she is the 19th Black transgender woman to be killed, and the third killed in Kansas City, specifically.
The Human Rights Campaign also estimates murders of trans people reached a high in 2017, and in 2018 approximately at least 26 trans people were killed, the vast majority of them being trans women of color.


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Doctors say Gaza epidemic would be preventable injustice “rooted in decades of Israeli oppression.” Read more about Johns Hopkins COVID-19 map faulted for erasing Palestinians
Israel seizes materials for a West Bank field clinic during epidemic. Read more about Palestinians face two enemies: occupation and pandemic
After smear campaign, lawyer with children’s rights group barred from addressing UN Security Council. Read more about Belgium abets Israel's crimes by silencing its critics
Fatou Bensouda has left open the possibility that judges will reject a full investigation. Read more about Will ICC pierce Israel's bubble of impunity?
Chief prosecutor’s annual update on preliminary examination contains “unwarranted and disturbing omissions,” rights groups state. Read more about Is ICC about to slam the door on justice for Palestine?
Human rights organization slams court decision: “extraordinarily dangerous precedent.” Read more about Israel’s high court orders expulsion of Omar Shakir
Israeli government report attempts to smear The Electronic Intifada. Read more about New Israeli report lies about anti-Semitism
Israeli forces raid offices of human rights defenders. Read more about Prisoners hunger strike as Israel reneges on pledges
Extreme ruling “undermines the most basic principles of universal humanity,” rights group says. Read more about Israeli court approves use of Palestinian bodies as bargaining chips
Calls to murder director of Al-Haq posted on Israeli government-run Facebook page. Read more about Israel incites death threats against human rights defenders
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Originally Posted by Drache
People have started calling me the unofficial Aftermath Kraken spokesperson lol! I took the Kraken out to a local milsim and it got good reviews from everyone who handled it. One guy with a TM AK was skeptical until he slapped in one of his TM AK Mags and fired off a few rounds.
It is the perfect cheap starter gun for people getting into the sport without spending the huge amounts of money. It out shot a stock TM AK for distance and accuracy as well as a CA M14 Match.
It one of those guns you can buy and play with while saving up for a name brand. So far mine has been through over 5000 rounds and the only problem was it dropped 5fps which is to be expected since a new spring loses some fps with regular use.
out ranged and better accuracy than TM?
thats crazy talk!


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that computer software
is inherently functional in respects that other
The current version of the Section 199 Treasury regulations starts with the general provision that DPGR includes, among other things, gross receipts "derived from the lease, rental, license, sale, exchange, or other disposition of computer software
" that is produced by the taxpayer in whole or in significant part within the United States.
Two recent decisions from the Federal Circuit affirm that not all computer software
patents are doomed, at least in the mind of the Federal Circuit.
The costs of developing computer software
(whether or not the particular software is patented or copyrighted) in many respects so closely resemble the kind of research and experimental expenditures that fall within the purview of [Sec.
1.199-3(i)(6)(v), a bank produces computer software
within the United States that enables customers to receive online banking services for a fee.
* Spending in the professional, scientific and technical services sector for ICT equipment and computer software
totaled $27 billion in 2008.
announced today that its premier tenant-screening solution, the ONLINE Rental Exchange, is now integrated with Simply Computer Software
, Inc.'s affordable housing management program called "FHA Software." This integration allows FHA Software users to view an instant credit risk decision, and run criminal background checks and eviction searches all from within FHA Software.
EVIDENCE emerged yesterday suggesting a Chinook helicopter crash which wiped out much of Northern Ireland's intelligence community could have been caused by faulty computer software
EVIDENCE has emerged suggesting a Chinook helicopter crash which wiped out much of Northern Ireland's intelligence community could have been caused by faulty computer software
NEW evidence has emerged that faulty computer software
could have led to a Chinook helicopter crash which killed 25 of Britain's top intelligence experts, it was reported.
Dubai: India's electronics and computer software
and services exports grew by 27.56 per cent in the fin-ancial year that ended on March 31, 2008, over the previous year, an Indian trade official has said.


Virtualize your proprietary hardware and create a more agile environment. Data center transformation helps the bottom-line and streamlines your business.
James Vogeltanz talks about the Xeon processors cloud orchestration capabilities.
Intel's NUC and Compute Stick are revolutionizing the industry's ideas about compute devices.
Optimize your software with Xeon Phi to gain significant performance without havnig to learn a new programming paradigm or new programming languages.


The Bear Mattress ranks No. 1 in our ratings of the Best Memory Foam Mattresses of 2021. Helix Mattress Vs Tuft And Needle
Here are the current top mattresses of 2021.
Entering 2021, the amount of mattress brands available can be overwhelming.
The below brands have been the top mattresses of 2021 thus far.
The brands are listed in no particular order.
So, if you’re searching for the best mattress available, you can’t go wrong with any of the following brands. If you are looking to buy a mattress online, you can’t go wrong with any of these brands.
- Puffy mattress has become the favorite for many people, who claim it to be the most comfortable mattress available. They often compare it to like sleeping on a cloud.
- Many with back pain claim to have relief, due to Puffy’s Adaptive Flexible Cloud Technology.
- With Puffy, you receive both a 101 night sleep trial, alongside a lifetime warranty.
- One of the highest quality mattresses for your money. Puffy is made to order, and ships in 2 days.
- Puffys’ first layer is CertiPUR-US Certified, and is made of Cooling Cloud Relief Foam. The Cooling Cloud Relief foam layer is the layer that gives you the “sleeping on a cloud” sensation. The bottom layer is comprised of a Firm Core Support Foam, which is similarly CertiPUR-US Certified. Helix Mattress Vs Tuft And Needle
- Another terrific mattress choice, is the ever popular DreamCloud mattress.
- Dreamcloud has great contour and bounce, thanks to its natural latex coat.
- Some of the best features of the Dreamcloud mattress, is its use of flax yarns and cashmere blends; along with stitched handles across the sides.
- Comes with a 365 night sleep trial, compared to 100 nights for most brands.
- Pocket Coil Technology allows for cheaper manufacturing costs, and the savings are based to you as the buyer. Helix Mattress Vs Tuft And Needle
- Purple Mattress is one of the most popular brands available.
- The Purple mattress is made from hyper-elastic polymer, which differs from many other mattresses, usually made from memory foam.
- Purple mattress feels different compared to any other brand on the market.
- Enables you to sleep cool throughout the night, all while having a really responsive motion transfer.
- Purple mattress includes a 10 year limited warranty.
- The top layer consists of a hyper-elastic polymer gel grid. The bottom layer is comprised of polyurethane foam. Helix Mattress Vs Tuft And Needle
- Nolah’s flagship product, the Signature uses proprietary AirFoam”, along with high density polyfoam for a soft but supportive feel.
- The Nolah Signature 12 is a luxury flippable all-foam mattress that’s medium-firm on one side and medium-soft on the other.
- If you have a partner, and are easily disturbed during the night, the Nolah Signature would be a great choice, due to how well it handles motion transfer.
- Every Nolah mattress comes with a 120 night sleep trial, and free shipping. Helix Mattress Vs Tuft And Needle
- Sweetnight sells a full range of mattresses and pillows, most of which feature gel memory foam as a main feature.
- Some Sweetnight products can be purchased on Amazon, but not all of their products, or mattress sizes.
- Sweetnight has 5 mattress models to choose from, both memory foam and hybrid.
- Sweetnight offers a 100 night sleep trial, as well as free shipping for all of its mattress models.
- Available in both memory foam and hybrid.You can purchase Sweetnight in a hybrid or memory foam model. Helix Mattress Vs Tuft And Needle
- Nectar Sleep has been voted Best Memory Foam mattress, and is one of the fastest growing companies in the sleep industry.
- Having optimal levels of firmness, coolness, breathability, and comfort with their mattresses, Nectar Sleep guarantees ideal sleep.
- Nectar is made of high quality all foam, and uses multiple layers.
- It is great for light to medium weight side and back sleepers, looking pressure relief and overall comfort and support.
- Nectar offers a Forever warranty on all of their mattresses, as well as gives you a full year to test them out. Helix Mattress Vs Tuft And Needle
- Awara is a hybrid mattress model, combining innerspring support with a significant comfort system.
- A latex hybrid, the Awara Mattress pairs a 4-inch layer of natural, Rainforest Alliance-certified Dunlop latex with a 9-inch support core of pocketed coils.
- Rainforest Alliance certified, the latex of the Awara meets criteria on economic, social, and environmental sustainability and impact.
- It is also certified by GOLS (Global Organic Latex Standard), which indicates that the latex is organic. The plush Euro top cover is composed of New Zealand wool and organic cotton. Helix Mattress Vs Tuft And Needle
- The The Saatva Classic mattress offers all the features side sleepers need without sacrificing the decadence of a luxury brand.Saatva Classic mattress offers all the features side sleepers need without sacrificing the decadence of a luxury brand.
- Fully customizable, the Saatva Classic offers 3 firmness level, sizes and heights. It is also made from only high-quality, eco-friendly materials.
- Saatva also has excellent customer service. They make the process stress-free by providing at-home setup for your new mattress and removal of your old mattress at no extra charge.
- Comes with a 180 night sleep trial, and free shipping.
- The Idle Sleep Hybrid is available in two firmness options: “medium,” which measures six out of 10 on the firmness scale, and “luxury firm,” which measures at an 8 out of 10.
- Customers may also opt for a mattress that measures “medium” on one side and “luxury firm” on the other.
- Idle Sleeps most popular model, the Idle Hybrid blends memory foam and pocketed coils, for just the right amount of bounce.
- Idle Sleep has the longest sleep trial in the industry at 18 MONTHS. Every mattress also comes with a lifetime warranty. Helix Mattress Vs Tuft And Needle
Support and Comfort
Comfort and support are two of the biggest aspects when looking to buy a new mattress.
There are a number of things which should take place when you lie down.
For one, you want the contour and sinkage to be correct, for proper spine alignment.
You will want adequate support while you sleep through out the night.
Proper alignment of the body while you sleep is critical.
This is important to make sure you are reducing neck and back pain, as well as have a healthy sleep.
While not only supporting your body, a mattress needs to be a good source of stress relief.
Your stress points are what actually cause you to thrash during the evening.
Memory foam mattresses are a great option for being both supportive and comfortable. Helix Mattress Vs Tuft And Needle
Should You Choose Firm or Soft?
This is much like when we speak on comfort and support.
Just like a mattress shouldn’t be too hard, it should never be too soft.
What you want in a mattress, is one that ensures your spine and body stay in a neutral position.
Too much rigidity in a mattress will irritate the stress variables, making pinched nerves, in addition to the blood to stop flowing.
If you find that a lot of the times you awake, with the sensation of having pins and needles in your body, this is a sign of a mattress that is too rigid.
If your mattress is too soft, you chance having a sagging effect, much like a hammock.
When this happens, you can find yourself with back pain, due to your spine not being correctly aligned.
This is why it’s imperative to find a mattress that is close to neutral.
What you want is a mattress that is soft in all the right places, but not too soft overall.
You likewise want a mattress that has some firmness to it; just not too rigid. Helix Mattress Vs Tuft And Needle
One feature most don’t think about when shopping for a new mattress, is the fever neutrality, or ‘breathability’ of a mattress.
Possibly the most effective attribute of memory foam mattresses now, is their capacity to be able to conform to your body.
Irregardless, foam mattresses which have diminished layers are well known to cause what’s known as”the oven result”.
Basically meaning the mattress catches and holds your body heat.
The good news is, with advanced technology, most mattress brands have a way to prevent this.
The goal of every mattress company is to offer breathability, allow you to sleep cool and also provide warmth where it should.
As with all things, some companies are better than others at doing this. Helix Mattress Vs Tuft And Needle
It’s not a huge surprise, that if you’re in the market for a high quality mattress, it normally comes with a nice price tag.
Since a mattress is a big investment, you certainly want to find one that will last for years to come.
It is crucial to discover a mattress that’s been well developed, with a lifetime span in mind.
Since you’re going to invest in a new mattress, make sure to pay attention to warranties. Find a brand with at least a 10 year warranty. Helix Mattress Vs Tuft And Needle
Quick Links to Other Top Mattresses: