Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In Vitro and In Vivo

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.

Chris Zhiyi Zhang^1,2, Yinghua Pan³, Yun Cao^1,2, Paul B. S. Lai⁴, Lili Liu^1,2, George Gong Chen^4*, Jingping Yun^1,2*

1 State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China, 2 Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China, 3 Department of Rheumatology and Immunology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, 4 Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.

Citation: Zhang CZ, Pan Y, Cao Y, Lai PBS, Liu L, et al. (2012) Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In Vitro and In Vivo. PLoS ONE 7(6): e39870. doi:10.1371/journal.pone.0039870

Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America

Received: April 10, 2012; Accepted: May 28, 2012; Published: June 28, 2012

Copyright: ? 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81172345 and No. 30973506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: yunjp@mail.sysu.edu.cn (JY); gchen@cuhk.edu.hk (GGC)

Introduction?Top

Liver cancer is the fifth most common cancer worldwide and the third most common cause of death from cancer [1]. More than 75% of new cases are diagnosed in developing countries; however, incidence is increasing in economically developed regions, including Japan, Western Europe, and the United States [2], [3]. Although surgical resection and liver transplant are the two major therapeutic options with curative potential, surgery is only feasible for about 20% of liver cancer cases since patients are most often diagnosed at an advanced stage [4], [5]. To date, chemotherapy for liver cancer is not satisfactory and the long-term survival of liver cancer patients is still poor [4], [6]. Therefore, developing novel and effective therapeutic strategies for liver cancer is of great need and significance.

Histone deacetylase inhibitors (HDACi) are currently a major focus of interest as antineoplastic agents [7], [8]. HDACi is a class of agents that function via blocking histone deacetylation, thereby modifying chromatin structure and gene transcription [9]. Particularly, HDACi inhibit the acetylation of lysine residues at the histone N-terminal tail which results in loosening the association of histones with DNA, thereby allowing the expression of genes related to tumor suppression [10]. Understanding the association between HDAC activities and various cancers led many researchers to consider HDAC inhibitors as potent agents that can interfere with cancer cell proliferation and/or survival through the modulation of cell cycle progression, differentiation, or by promoting cell death. For example, Kim et al. reported that CG0006 exposure in breast cancer cell resulted in cell death via down-regulation HDAC6 [11]. Bommi et al. demonstrated that sodium butyrate induced apoptosis in cancer cells by transcriptional downregulation of BMI1 [12].

Although HDACi alone may be clinically useful, they will most likely be of value in combination with other antitumor agents. SAHA has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma and other HDACi are now undergoing Phase I/II clinical trials as a single agent or in combination with other agents [13], [14]. Accumulating reports have been indicated the synergistic effect on lethality of combination of HDACi and other chemotherapeutic agents. Kretzner et al. showed that combination of HDACi and Aki enhanced lymphoma cell death through repression of c-Myc, hTERT, and microRNA levels [15]. Nguyen et al. reported that coadministration of HDACi synergistically increased KW-2449 lethality resulting from inactivation of Bcr/Abl [16]. Lately, a phase II study revealed that treatment of vorinostat combined with tamoxifen significantly prolonged the survival of patients with breast cancer [17]. However, such a synergistic effect has rarely been demonstrated in liver cancer.

Recently, we have reported that Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, exhibited anticancer activity towards liver cancer [18]. In the present study, we showed that (a) DHA induced apoptosis via downregulating ERK phosphorylation, which was further confirmed by the data that the inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis, (b) HDACi in vitro remarkably enhanced DHA-induced cell death, accompanying with reduction of mitochondria membrane potential, release of cytochrome c into cytoplasm, increase of p53 and Bak, and decreases of Mcl-1 and p-ERK, (c) the combination of HDACi and DHA in vivo significantly halted the growth of liver cancer tumor xenograft. Our data may suggest the combination of HDACi and DHA as a promising strategy for liver cancer chemotherapy.

Materials and Methods?Top

Cell culture

Human liver cancer cell lines (Hep G2 and PLC/PRF/5) were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD) containing 10% fetal bovine serum (FBS), 100 mg/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO₂ and 95% air at 37?C.

Antibodies and reagents

Antibodies for Mcl-1, PARP, Bak, and Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for caspase 3, p38, p-p38, ERK, p-ERK, JNK, and p-JNK were provided by Cell Signaling (Danvers, MA). Dihydroartemisinin (DHA, dissolved in DMSO), sodium butyrate (NaB, dissolved in H₂O), suberoylanilide hydroxamic acid (SAHA, dissolved in DMSO) and p-ERK inhibitor (PD98059, dissolved in DMSO) were purchased from Sigma (St. Louis, MO).

MTT

Cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-lium bromide (MTT) assay. Briefly, 8?10³ of cells were seeded into 96-well plates for 24 h, followed by incubation with various doses of DHA for indicated time. After adding 100 ?l/well of MTT solution, the cells were incubated for another 2 h. Supernatants were then removed and the formazan crystals were dissolved in 100 ?l/well DMSO. The absorbance at 570/630 nm of each sample was measured using multilabel plate reader (PerkinElmer). Three independent experiments were performed.

Colony formation

One hundred of cells were seeded into 6-well plates, and cultured for 7 d. And then the medium was replaced by fresh one containing DHA. After being incubated for another 7 d, colony formed by liver cancer cells was stained with 0.05% crystal violet (Sigma, St. Louis, MO) for 8 min. The number of colony was then quantified.

Western blot

Cell lysates were boiled with 6x sodium dodecyl sulfate (SDS) loading buffer and then fractionated by SDS-PAGE. The proteins were transferred to PVDF membrane which was then incubated with a primary specific antibody in 5% of non-fat milk, followed by a horse radish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit second antibodies. ECL detection reagent (Amersham Life Science, Piscataway, NJ) was used to demonstrate the results.

Annexin V/PI assay

Apoptosis was assessed using Annexin V-PI double staining. After treatments, cells were trypsinized, and stained with 0.5 mg/ml Annexin V in binding buffer (10 mM HEPES free acid, 0.14 M NaCl, and 2.5 mM CaCl₂) for 30 min. Afterward, PI (5 ?g/mL final concentration) was added and incubated for another 15 min. Cells were applied to a flow cytometer for data collection.

TUNEL assay

Apoptosis assay was performed using Apo-Direct TUNEL Assay kit (Millipore). Cells were harvested and fixed in 4% PFA for 60 min at 4?C, followed by a second fixation in 70% (v/v) ethanol overnight at ?20?C. Cells were then treated with various reagents for a designed period according to the manufacture's instruction. Finally, cells were analyzed by flow cytometry using FACS Vantage machine (Becton Dickinson). The Cell Quest software (Verity Software House) was used to analyze the data.

In situ cell death detection

Labeling of fragmented DNA to assess apoptosis was performed with TUNEL staining (green fluorescence), using In Situ Cell Death Detection Kit (Roche, LA), as described in our previous study [19].

Measurement of caspase 3 activity

The activity of caspase 3 induced by DHA treatment was determined by the Caspase-3 Activity Assay Kit (Merck, Darmstadt).

Measurement of mitochondrial membrane potential (??_m) by flow cytometry

Forty nM of DioC6 (Sigma?Aldrich, MO) were incubated with treated cells at indicated time points for 15 min at 37?C. The harvested cells were washed with ice-cold PBS and analyzed by flow cytometry using Becton Dickinson FACS Vantage machine (Becton Dickinson, NJ). Cells with low ??_m were presented as a percentage of the total cell population. The CellQuest software (Verity Software House) was used to analyze the data.

Animal studies

All animal experiments were conducted according to relevant national and international guidelines and have been approved by the Institute Research Medical Ethics Committee of Sun Yat-Sen University Cancer Center. 1?10⁷ of Hep G2 cells were suspended in sterile PBS and injected subcutaneously into the right flank of the mice. Mice were checked daily for xenograft/tumor development. Mice were randomized into three groups of 6 mice/ group. DHA (5 mg/kg mouse body weight) was given to the ?DHA? group, SAHA (1.5 mg/kg mouse body weight) was given to the ?SAHA? group, combination of SAHA and DHA was given to ?DHA+SAHA? group, once daily for five consecutive days per week for 24 d. The DMSO group received an equal volume of solvent control. After treatment at various time intervals, mouse body weight and tumor size were measured. Finally, tumors were excised, weighed and fixed in 4% of PFA. Paraffin-embedded tissues were then sectioned at 4 nm and ready for H&E staining and TUNEL assay.

Immunohistochemistry

Formalin-fixed and paraffin-embedded liver cancer sections with a thickness of 4 ?m were dewaxed in xylene and graded alcohols, hydrated, and washed in phosphatebuffered saline (PBS). After pretreatment in a microwave oven, endogenous peroxidase was inhibited by 3% hydrogen peroxide in methanol for 20 min, followed by avidin-biotin blocking using a biotin-blocking kit (DAKO, Germany). Slides were then incubated with antibodies for 4 h in a moist chamber at room temperature, washed in PBS, and incubated with biotinylated goat anti-rabbit/mouse antibodies. Slides were developed with the Dako Liquid 3, '3-diaminobenzidine tetrahydrochloride (DAB) +Substrate Chromogen System and counterstained with hematoxylin.

Statistical analysis

Difference between groups was determined for statistical significance using one-way ANOVA or Student's t-test. All P-values are two-sided and P<0.05 was considered as statistically significant. All statistical calculations were performed with the SPSS software (SPSS, Inc., Chicago, IL). The data were presented as mean?SD from at least three independent experiments.

Results?Top

Activations of MAP kinases were involved in DHA-induced apoptosis

DHA has been demonstrated to induce cell death in human cancers [20], [21]. We first assessed DHA-induced apoptosis in liver cancer cell lines, using Annexin V assay. Results indicated that percentage of Annexin V-positive cells were dramatically increased upon DHA treatment (Fig. 1A), suggesting DHA being a potent apoptosis inducer in liver cancer cells. Compared to the control, following exposure of 10 ?M DHA for 24 h, the percentage of apoptotic cells was remarkably increased from 5.3% and 4.9% to 16.6% and 13.5%, respectively in Hep G2 and PLC/PRF/5 cells (Fig. 1B).

Figure 1. Activations of MAP kinases were involved in DHA-induced apoptosis.

A. DHA induced apoptosis in liver cancer cells. Cells treated with either DMSO or 10 ?M DHA for 24 and 48 h were stained with both Annexin V and Propidium Iodide (PI) for 45 min. Apoptosis induced by DHA was then assessed by flow cytometer analysis. B. The percentage of apoptotic cells were shown, after quantitative analysis of PI/Annexin V assay. Data are presented as mean?SD of three independent experiments. *P<0.05, versus the DMSO group. C. MAP kinases were activated by DHA. Cells were treated with 10 ?M DHA for indicated time. The phosphorylation of p38, ERK and JNK was determined. D. Quantitative data from three independent experiments were shown to indicate the relative expression of p-p38, p-JNK, and p-ERK.

doi:10.1371/journal.pone.0039870.g001

Apoptosis induction usually associates with activation of MAP kinases. A time-course analysis was performed on the phosphorylation levels of three MAP kinase members, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 (Fig. 1C). The protein levels of all 3 MAP kinases remained unchanged. However, p38 phosphorylation was increased after DHA treatment in both tested cells. The level of JNK phosphorylation remained the same as control in Hep G2 cells, but was markedly increased in PLC/PRF/5 cells (Fig. 1D). The levels of p-ERK appeared decreasing in both liver cancer cells treated with DHA. These data may suggest that inactivation of ERK contributes to DHA-induced apoptosis.

Inhibition of ERK phosphorylation was attributed to DHA-induced apoptosis in liver cancer cells

To test our assumption that inactivation of ERK was involved in DHA-induced apoptosis, we pretreated cells with PD98059, an inhibitor of ERK phosphorylation. Firstly, the cytotoxicity of PD98059 was tested. Results indicated that PD98059 alone did not cause significant apoptosis in both cells (data not shown). We next determined the effect of PD98059 on DHA-induced cell growth attenuation. As indicated by MTT result, PD98059 significantly reduced liver cancer cell viabilities, compared with DHA groups (Fig. 2A).

Figure 2. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis. A.

PD98059, an inhibitor of ERK phosphorylation, enhanced DHA-induced decrease of cell viability. Cells were pretreated with 10 ?M PD98059 for 2 h, and then incubated with 10 ?M DHA for another 24 h. The residual cell viability was determined by MTT assay. Data are mean ? SD of three independent experiments, *P<0.05. B. PD98059 treatment increased the production of apoptotic body. Cells pretreated with 10 ?M PD98059 for 2 h, and then incubated with 10 ?M DHA for another 24 h were stained with Hoechst 33342 dye. DNA fragmentation (indicated by asterisks) and nuclear condensation (denoted by arrows) were observed under a fluorescence microscope. C. PD98059 treatment enhanced DHA-induced apoptosis. TUNEL assay was performed to determine apoptosis. The number of apoptotic cells was determined and the percentage was indicated by histogram. *P<0.05. D. PD98059 plus DHA treatment led to cleavages of PARP and caspase 3. Proteins collected from cells treated with PD98059 and DHA for 24 h were subjected to western blot to detect the cleavages of PARP and caspase 3. Actin was used as a loading control.

doi:10.1371/journal.pone.0039870.g002

Next, we examined whether PD98059 treatment enhanced DHA-induced cell growth inhibition through inducing apoptosis. We assessed DHA-induced apoptosis in liver cancer cells pretreated with 10 ?M PD98059 for 2 h by Hoechst 33342 staining. Results revealed more cells with characteristic features including chromatin condensation and apoptotic body presented in PD98059-pretreated cells (Fig. 2B). This was further confirmed by TUNEL assays showing that the percentages of TUNEL-positive cells were increased in liver cancer cells treated with both PD98059 and DHA (Fig. 2C). Moreover, levels of cleaved PARP and cleaved caspase 3 were noticeably increased by the ERK inhibitor in the 2 liver cancer cell lines (Fig. 2D). These findings suggest that DHA-induced apoptosis may be related to ERK phosphorylation.

HDACi facilitated DHA-induced apoptosis in liver cancer cells

In view of that HDACi is capable of enhancing the lethal effect of chemotherapeutic agents [22], [23], we intended to examine whether DHA combined with HDACi resulted in more cell death in liver cancer. NaB and SAHA were used in MTT analysis. According to the results, combination of DHA and HDACi significantly reduced cell viabilities in liver cancer cells, compared to treatment with single agent (Fig. 3A). The increased cytotoxicity of combination of DHA and HDACi was also determined by colony formation assay. Cells in DMSO groups formed a number of visible colonies in 15 d. The number of colony formed by cells cultured with both DHA and HDACi was significantly less than that with DHA alone (Fig. 3B).

Figure 3. HDACi facilitated DHA-induced apoptosis. A.

Combination treatment with HDACi and DHA increased cell death. Cells were treated with 4 mM NaB, 1.25 ?M SAHA, 10 ?M DHA or combination of NaB/SAHA and DHA for 24 h. Cell viabilities were measured by MTT assay. B. The inhibitory effect of HDACi and DHA in combination on liver cancer cell growth was further confirmed by colony formation assay. One hundred of cells were seeded into 6-well plates for 7 d, and then cultured with either HDACi or DHA for another 7 d. Colonies were stained with 0.05% crystal violet. The number of colony in each well was counted and statistical analysis was performed. Data are presented as mean ? SD of three independent experiments. C. The effect of HDACi on DHA-induced apoptosis was measured by TUNEL assay, using in situ cell death detection kit. Hep G2 cells treated as described in A were subjected to TUNEL assay. Apoptotic cells were observed under fluorescent microscope. D. The effect of HDACi and DHA in combination was further confirmed by TUNEL assay, using flow cytometry. Percentage of apoptotic cells was calculated. E. Caspase 3 activation was involved in HDACi-mediated apoptosis in cells treated with DHA. The activity of caspase 3 in cells treated as described in A was determined and the relevant change was shown. For A, B, D and E, *P<0.05, **P<0.01, versus the DHA group.

doi:10.1371/journal.pone.0039870.g003

Next we determined the pro-apoptotic activity of combined treatment with DHA and HDACi. DHA treatment potently induced apoptosis in liver cancer cells, but more apoptosis was induced by the combined treatment with both agents, as shown by TUNEL assays indicating a noticeable increase in TUNEL-positive cells (Fig. 3C). Statistically, DHA in combination with NaB or SAHA increased apoptotic cells by 1.9 or 2.8 fold, respectively in Hep G2 cells, and by 3.0 or 3.5 fold respectively in PLC/PRF/5 cells (Fig. 3D). In line with the increased apoptosis, caspase 3 activity was higher in cells treated with both DHA and HDACi (Fig. 3E).

Release of cytochrome c into cytoplasm and downregulation of Mcl-1 and p-ERK contributed to apoptosis caused by the combined treatment with HDACi and DHA

We have previously demonstrated that DHA-induced apoptosis associated with Mcl-1 degradation and Bak activation [18]. We next examined whether Mcl-1 and Bak were involved in HDACi-mediated enrichment of apoptosis in DHA-treated cells. As indicated in results of western blot, Mcl-1 was dramatically decreased, whereas Bak was markedly increased in Hep G2 cells treated with both DHA and NaB/SAHA. The alterations of Mcl-1 and Bak in PLC/PRF/5 cells shared a similar trend with those in Hep G2 cells (Fig. 4A). In addition, wild-type p53 in Hep G2 cells were upregulated, while mutant p53 in PLC/PRF/5 cells were hardly affected, following the treatment (Fig. 4A).

Figure 4. HDACi exposure in DHA-treated cells enhanced decreases of Mcl-1 and p-ERK. A.

Combination treatment with HDACi and DHA resulted in downregulation of Mcl-1 and upregulation of Bak. liver cancer cells were exposed to 4 mM NaB, 1.25 ?M SAHA, 10 ?M DHA or combination of NaB/SAHA and DHA for 24 h. Expression of Mcl-1, Bak and cleaved PARP was examined by western blot. Upper panel: a representative result was shown. Bottom panel: the relative expression of Mcl-1 and Bak normalized to Actin was indicated by histogram. B. The expression of p-ERK was reduced in cells treated with HDACi and DHA. Proteins collected from liver cancer cells treated with SAHA, DHA or the combined drugs were subjected to western blot to examine p-ERK expression. Upper panel: a representative result was presented. Bottom panel: the relative expression of p-ERK/ERK was shown. C. Reduction of mitochondrial membrane potential (MMP) was induced in HDACi/DHA-treated cells. Hep G2 and PLC/PRF/5 cells were treated as described in A. The MMP collapse (??_mL) was measured by flow cytometry after staining the cells with DioC6 and quantitative analysis of ??_m was shown. The data represented mean?SD of three independent experiments. D. Cytochrome c was released to cytosol in treated cells. Cells were treated as described in A. Fractions of cytosol were isolated to examine the distribution of cytochrome c. ?-Actin was used as the marker for cytosol. E. HDACi pretreatment sensitized cells to DHA-induced apoptosis. liver cancer cells pretreated with 4 mM NaB or 1.25 ?M SAHA for 2 h were further exposed to 10 ?M DHA for another 24 h. TUNEL assays were performed to determine apoptosis. The percentage of TUNEL-positive cells was shown. For B, C and E, *P<0.05, versus the DHA group.

doi:10.1371/journal.pone.0039870.g004

In light of emerging data that ERK phosphorylation was inhibited in DHA-treated and HDACi-treated cells. We next examined the level of phosphorylated ERK. Results showed a rapid decrease of p-ERK in liver cancer cells treated with both DHA and SAHA, especially in PLC/PRF/5 cells (Fig. 4B).

Since DHA-induced apoptosis was attributed to the depolarization of mitochondrial outer membrane [18], we next examined the reduction of mitochondrial membrane potential (MMP). Results showed that the combined treatment remarkably lowered the mitochondrial transmembrane potential (Fig. 4C), followed by an obvious release of cytochrome c from mitochondria to cytoplasm (Fig. 4D).

As shown in our previous study, HDACi pretreatment sensitized liver cancer cells to etoposide [22]. We pretreated cells with NaB or SAHA, and then assessed the resulting apoptosis by TUNEL assays. Compared to those of unpretreated cells, percentages of TUNEL-positive cells in HDACi-pretreated cells were significantly increased (Fig. 4E).

SAHA enhanced antitumor effect of DHA on Hep G2 xenograft tumor in mice

Having demonstrated the ability of SAHA to enhance DHA-mediated cell death in vitro, we further determined the synergetic effect of SAHA and DHA in vivo. Hep G2 cells were subcutaneously injected in nude mice to establish tumor xenograft. Nude mice bearing tumor xenografts were dosed with DHA (5 mg/kg/Bid) and/or SAHA (1.5 mg/kg/Bid) daily for 24 days. The treatment did not appear to have a noticeable effect on body weight in mice. On average, the combination therapy inhibited liver cancer tumor growth by more than 44.7% while the single agent treatment with either DHA or SAHA only inhibited the tumor growth by 17.6% and 4.6%, respectively (Fig. 5A). On Day 24, mice were sacrificed and the tumor weights were measured. As expected, the combination of HDACi and DHA significantly reduced the weights of xenograft tumor, compared with DHA-only groups (Fig. 5B). These data indicated that the combination treatment generated a greater anti-proliferative effect and cytotoxicity than either single agent alone in liver cancer xenografts in vivo.

Figure 5. SAHA enhanced antitumor effect of DHA on Hep G2 xenograft tumor in mice. A.

Combination of SAHA and DHA noticeably halted the growth of Hep G2 xenograft tumor. Nude mice were inoculated with 1?10⁷ of Hep G2 cells. After the formed tumor was palpable, mice were randomly divided into four groups. DHA (5 mg/kg mouse body weight) was given to the ?DHA? group, SAHA (1 mg/kg mouse body weight) was given to the ?SAHA? group, combination of SAHA and DHA was given to 'DHA+SAHA' group, once daily for five consecutive days per week for 24 d. The tumor volumes were calculated every two days. Six xenografts were performed in each group. Data are mean?SD, *P<0.05, versus the DMSO group. B. Combination of SAHA and DHA treatments resulted in a dramatic decline of tumor weight. On day 24, mice were sacrificed, and the tumor weights were measured. C. Apoptosis was induced in vivo. Tumors were sectioned and apoptosis was determined using in situ cell death detection kit. Apoptotic cells were observed under fluorescent microscope. D. SAHA significantly increased apoptosis in DHA-treated mice. Percentages of apoptotic cells were measured by counting the number of green cells under five random fields.

doi:10.1371/journal.pone.0039870.g005

In order to test whether HDACi enhanced the lethal effect of DHA via increasing apoptosis, tumor tissues were sectioned and subjected to in situ cell death detection (Fig. 5C). Results showed that the proportion of apoptotic cells was significantly increased from 8.6?2.4% in DHA group to 17.7?3.3% in combined treatment group (Fig. 5D). In addition, we examined the histology of tumors after the treatment using H&E staining. Tumors from control group showed typical histological appearance of liver cancer (Fig. 6). The sections of DHA-treated tumors showed that cancer cells were markedly decreased, with signs of apoptosis, infiltration of inflammatory cells and fibrosis. In the combined treated group, apoptotic regions and extensive necrosis with infiltration of phagocytic cells could be observed fairly often.

Figure 6. Decreased expression of Mcl-1 and increased levels of active PARP and cleaved caspase 3 were recorded in HDACi/DHA-treated mice.

SAHA enlarged the apoptotic region caused by DHA treatment in Hep G2 xenograft tumor. Tumors were excised and subjected to H&E staining for determination of pathological evaluation. On the other hand, tissues of xenografts were subjected to immunochemistry to detect the expression of Ki-67, p53, Mcl-1, p-ERK, and active PARP. Original magnification ?400.

doi:10.1371/journal.pone.0039870.g006

In addition, we performed immunohistochemistry to detect the proteins involved in HDACi/DHA-induced apoptosis (Fig. 6). Decreased expression of Ki-67 indicated the reduction of cell proliferation and likely enhanced cell death. Detectable difference in p53 expression was observed. Striking increase of active PARP, as well as a predominant decline of Mcl-1 and p-ERK, was present in HDACi/DHA-treated xenograft. Taken together, these data indicated that HDACi were able to significantly augment DHA-mediated antitumor effects.

Discussion?Top

Recent studies suggest that HDACi including NaB and SAHA interact synergistically with cytotoxic agents, such as fludarabine and etoposide, to dramatically increase mitochondrial injury and apoptosis in leukemic and epithelial cancer cells [24], [25]. The antitumorigenic properties of HDACi are especially notable probably due to the fact that their cytotoxic effects are usually specific to cancer cells but not to normal cells. However, when used as a single agent, HDACi might exhibit limited lethal activity towards liver cancer, which is evident in the present study showing that both NaB and SAHA at low doses are unable to induce significant growth inhibition in vitro and in vivo. However, when HDACi are used in combination with DHA, a derivative of artemisinin that is clinically used in malaria treatment with good toxicity profile [26], they can induce much more apoptosis, resulting in remarkable halt of tumor xenograft in nude mice. There is very limited information on HDACi in combination with other anti-tumor agents against liver cancer. Our data for the first time have demonstrated a synergic effect of DHA and HDACi in inhibition of liver cancer.

Many reports have demonstrated that the threshold of apoptosis in cancer cells can be controlled by the activities of multiple signal transduction pathways, one of which is Raf-MEK1/2-ERK1/2 pathway [27], [28]. This pathway is frequently dysregulated in neoplastic transformation, along with the c-Jun NH2-terminal kinase (JNK1/2) and p38 MAPK pathways [29]. It has also been implicated that activation of the ERK1/2 pathway is usually associated with survival but JNK1/2 and p38 MAPK pathway with apoptosis [30]. In our study, ERK1/2 phosphorylation was slightly inhibited by DHA treatment but strongly inhibited by the combined treatment with DHA and HDACi. In addition, using the ERK-specific inhibitor PD98059, we demonstrated that the activation of the ERK is antiapoptotic since the ERK inhibitor enhanced DHA-induced apoptosis in liver cancer cells.

A number of antiapoptotic effector proteins have been identified downstream of ERK1/2 signaling, including Bcl-xL and Mcl-1 [31], [32]. Alterations of both phosphorylated ERK and Mcl-1 frequently occurs in the same direction. Yuen et al. reported that silencing Ran may lead to deactivation of ERK and downregulation of Mcl-1 in cancer cells [33]. Reeves et al. showed that the activation of ERK and induction of Mcl-1 were observed in myeloid cells infected by human cytomegalovirus [34]. Calvi?o et al. reported treatement with lonidamine plus arsenic trioxide resulted in reductions of Mcl-1 and p-ERK [35]. In our previous study, Mcl-1 was downregulated in DHA-treated cells [ref]. Here we further showed a decrease of phosphorylated ERK in DHA-exposed liver cancer cells. Collectively, it seems that there is some certain correction between Mcl-1 and p-ERK: one protein could be regulated by the other. Interestingly, Konopleva et al. showed that MEK inhibitors such as PD0325901 and CI-1040, which are capable of inhibiting the activation of ERK, successfully suppressed Mcl-1 expression in Leukemia cells [36]. Booy et al. demonstrated that knockdown of ERK1 or inhibition of the ERK phosphorylation sufficiently inhibited EGF-mediated Mcl-1 upregulation [37]. Sun et al. showed that the overexpression of ERK partly reversed EPOX-induced Mcl-1 degradation in tumor cells [32]. However, the detailed mechanism through which ERK affects Mcl-1 expression requires further investigation. In view of that (a) a decrease of Mcl-1 is essential for the induction of apoptosis by diverse apoptotic stimuli caused by different types of chemotherapeutic agents; (b) deactivation of ERK may result in Mcl-1 degradation; and (c) the administration of both HDACi and DHA synergistically regulate ERK phosphorylation and Mcl-1 expression, the combination treatment with HDACi and DHA should have a great clinical potential in the improvement of liver cancer treatment.

In light of the findings that (a) DHA induced more cell death in liver cancer cells bearing wild-type p53, (b) HDACi can lead to upregulation of p53, we rationally assumed that the combined treatment with DHA and HDACi increased apoptosis probably via inducing p53 expression in Hep G2 cells. However, more evidence should be obtained to verify the assumption. On the other hand, cells with p53 mutants have been demonstrated to be less sensitive to DHA, but can significantly respond to HDACi treatment [38]. Therefore, if the combination of both agents is used to treat p53-mutated cells, the apoptosis induced is likely to be comparable with that in cells with wild-type p53. In fact, such an assumption is proved in our present experiment. This finding indicates that HDACi and DHA in combination can be applied to both p53-wide type and p53-mutated liver cancer with similar efficacy. This is of clinical significance since p53 mutants are presented in most of liver cancer cases.

According to our results that (a) in vitro data showed that combination of HDACi and DHA significantly reduced cell viability in both cells, and (b) in vivo data revealed a remarkable decrease of Ki-67, this combined treatment resulted in significant cell growth inhibition which may lead to the resulting antitumor effects. However, further study should be carried on to disclose the exact mechanism through which cell growth inhibition, in addiction to apoptosis, caused by combination of HDACi and DHA contributed to tumor inhibition.

In conclusion, our in vitro and in vivo data highlight that the combination treatment with HDACi and DHA has a synergic effect in the induction of liver cancer cell death, and this strategy can also reduce the dose of HDACi and thus it may circumvent the inherent toxicity. Mechanically, our study has demonstrated that the combination treatment can regulate ERK phosphorylation and Mcl-1 expression to induce apoptosis of liver cancer cells independent of p53 status.

Author Contributions?Top

Conceived and designed the experiments: JPY GGC CZYZ. Performed the experiments: CZYZ YHP YC LLL. Analyzed the data: JPY GGC CZYZ PBL. Wrote the paper: CZYZ YHP YC PBL JPY.

References?Top

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11. Kim HM, Kim CS, Lee JH, Jang SJ, Hwang JJ, et al. (2011) CG0006, a novel histone deacetylase inhibitor, induces breast cancer cell death via histone-acetylation and chaperone-disrupting pathways independent of ER status. Breast Cancer Res Treat 130: 365?375. Find this article online
12. Bommi PV, Dimri M, Sahasrabuddhe AA, Khandekar J, Dimri GP (2010) The polycomb group protein BMI1 is a transcriptional target of HDAC inhibitors. Cell Cycle 9: 2663?2673. Find this article online
13. Gould JJ, Kenney PA, Rieger-Christ KM, Silva Neto B, Wszolek MF, et al. (2010) Identification of tumor and invasion suppressor gene modulators in bladder cancer by different classes of histone deacetylase inhibitors using reverse phase protein arrays. J Urol 183: 2395?2402. Find this article online
14. Molife LR, Attard G, Fong PC, Karavasilis V, Reid AH, et al. (2010) Phase II, two-stage, single-arm trial of the histone deacetylase inhibitor (HDACi) romidepsin in metastatic castration-resistant prostate cancer (CRPC). Ann Oncol 21: 109?113. Find this article online
15. Kretzner L, Scuto A, Dino PM, Kowolik CM, Wu J, et al. (2011) Combining histone deacetylase inhibitor vorinostat with aurora kinase inhibitors enhances lymphoma cell killing with repression of c-Myc, hTERT, and microRNA levels. Cancer Res 71: 3912?3920. Find this article online
16. Nguyen T, Dai Y, Attkisson E, Kramer L, Jordan N, et al. (2011) HDAC inhibitors potentiate the activity of the BCR/ABL kinase inhibitor KW-2449 in imatinib-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo. Clin Cancer Res 17: 3219?3232. Find this article online
17. Munster PN, Thurn KT, Thomas S, Raha P, Lacevic M, et al. (2011) A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer. Br J Cancer 104: 1828?1835. Find this article online
18. Zhang CZ, Zhang H, Yun J, Chen GG, Lai PBS Dihydroartemisinin exhibits antitumor activity toward hepatocellular carcinoma in vitro and in vivo. Biochemical Pharmacology.
19. Zhang CZ, Chen GG, Merchant JL, Lai PB (2012) Interaction between ZBP-89 and p53 mutants and its contribution to effects of HDACi on hepatocellular carcinoma. Cell Cycle 11: 322?334. Find this article online
20. Chen T, Li M, Zhang R, Wang H (2009) Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy. J Cell Mol Med 13: 1358?1370. Find this article online
21. Lu JJ, Meng LH, Shankavaram UT, Zhu CH, Tong LJ, et al. (2010) Dihydroartemisinin accelerates c-MYC oncoprotein degradation and induces apoptosis in c-MYC-overexpressing tumor cells. Biochem Pharmacol 80: 22?30. Find this article online
22. Zhang CZ, Zhang HT, Chen GG, Lai PB (2011) Trichostatin A sensitizes HBx-expressing liver cancer cells to etoposide treatment. Apoptosis 16: 683?695. Find this article online
23. Wagner S, Roemer K (2005) Retinoblastoma protein is required for efficient colorectal carcinoma cell apoptosis by histone deacetylase inhibitors in the absence of p21Waf. Biochem Pharmacol 69: 1059?1067. Find this article online
24. Rosato RR, Almenara JA, Maggio SC, Coe S, Atadja P, et al. (2008) Role of histone deacetylase inhibitor-induced reactive oxygen species and DNA damage in LAQ-824/fludarabine antileukemic interactions. Mol Cancer Ther 7: 3285?3297. Find this article online
25. Dalgard CL, Van Quill KR, O'Brien JM (2008) Evaluation of the in vitro and in vivo antitumor activity of histone deacetylase inhibitors for the therapy of retinoblastoma. Clin Cancer Res 14: 3113?3123. Find this article online
26. Willoughby JA Sr, Sundar SN, Cheung M, Tin AS, Modiano J, et al. (2009) Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression. J Biol Chem 284: 2203?2213. Find this article online
27. Ehses JA, Pelech SL, Pederson RA, McIntosh CH (2002) Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway. J Biol Chem 277: 37088?37097. Find this article online
28. Jazirehi AR, Vega MI, Chatterjee D, Goodglick L, Bonavida B (2004) Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab. Cancer Res 64: 7117?7126. Find this article online
29. Aquilano K, Baldelli S, Rotilio G, Ciriolo MR (2009) trans-Resveratrol inhibits H2O2-induced adenocarcinoma gastric cells proliferation via inactivation of MEK1/2-ERK1/2-c-Jun signalling axis. Biochem Pharmacol 77: 337?347. Find this article online
30. Rahman MS, Yamasaki A, Yang J, Shan L, Halayko AJ, et al. (2006) IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways. J Immunol 177: 4064?4071. Find this article online
31. Sawatzky DA, Willoughby DA, Colville-Nash PR, Rossi AG (2006) The involvement of the apoptosis-modulating proteins ERK 1/2, Bcl-xL and Bax in the resolution of acute inflammation in vivo. Am J Pathol 168: 33?41. Find this article online
32. Sun HL, Tsai AC, Pan SL, Ding Q, Yamaguchi H, et al. (2009) EPOX inhibits angiogenesis by degradation of Mcl-1 through ERK inactivation. Clin Cancer Res 15: 4904?4914. Find this article online
33. Yuen HF, Chan KK, Grills C, Murray JT, Platt-Higgins A, et al. (2012) Ran Is a Potential Therapeutic Target for Cancer Cells with Molecular Changes Associated with Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK Pathways. Clin Cancer Res 18: 380?391. Find this article online
34. Reeves MB, Breidenstein A, Compton T (2012) Human cytomegalovirus activation of ERK and myeloid cell leukemia-1 protein correlates with survival of latently infected cells. Proc Natl Acad Sci U S A 109: 588?593. Find this article online
35. Calvino E, Estan MC, Simon GP, Sancho P, Boyano-Adanez Mdel C, et al. (2011) Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation. Biochem Pharmacol 82: 1619?1629. Find this article online
36. Konopleva M, Milella M, Ruvolo P, Watts JC, Ricciardi MR, et al. (2011) MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex. Leukemia.
37. Booy EP, Henson ES, Gibson SB (2011) Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer. Oncogene 30: 2367?2378. Find this article online
38. Li D, Marchenko ND, Moll UM (2011) SAHA shows preferential cytotoxicity in mutant p53 cancer cells by destabilizing mutant p53 through inhibition of the HDAC6-Hsp90 chaperone axis. Cell Death Differ 18: 1904?1913. Find this article online

Source: http://feeds.plos.org/~r/plosone/PLoSONE/~3/-VRmge5zWO8/info%3Adoi/10.1371/journal.pone.0039870

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ZapDUDE! ? Blog Archive ? Learning From ESPN Horse Racing

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Horse racing is one of the biggest spectator sports in existence today. Some people may define Horse racing as gambling, but in fact, it has always been a sport for the competitors.

When you take a look at history, you will find out that the people who first raced horses did not do so for money, people used to race horses because of pride. They wanted to prove that they are superior to their opponents. Do you want proof that horse racing is indeed a sport? Then you should take a look at the ESPN horse racing index.

Many people are surprised when they learn that ESPN even has a horse racing index. This just goes to show you that not many people think that horse racing is still a sport. The ESPN horse racing index, however, is proof to the fact that horse racing does have a place in the world of sports.

Just what can you learn from the ESPN horse racing index?

Of course, you can find news about horse racing in the ESPN horse racing index. These bits of news tell what goes on in the track and what you need to watch out for. This news can also help a gambler determine his pick. This is because of the fact that the news on ESPN horse racing index tells of the latest events which could significantly affect various races.

In the ESPN horse racing index, you can also find news regarding rising stars of the track. Through the ESPN horse racing index, you can keep an eye on these horses and try to see how they could affect you horse racing experience. The news in the ESPN horse racing index also shows recent events which could affect you. They show the condition of various horses. They report any injury which could affect race results drastically.

In the ESPN horse racing index, you can also find the results of various races. This makes knowing results very convenient for you. There are people who like betting on horse racing but they may not have time to go out to the track and watch the race. Some just go to the track to bet and wait for the results to be announced later. Through the ESPN horse racing index, you can know if you should go down to the track to collect your cash or if you should just stay at home and try to forget that you even betted.

Several links can also be found in the ESPN horse racing index. These links could link you to horse trainers who can help condition your horses for a race, or they could link you to a track and allow you to bet through the internet. There are also links which may lead to information concerning horse races which you might not find in the ESPN horse racing index.

All in all, the ESPN horse racing index is a great information source for those who are big fans of horse racing. The articles are well written and show the sports side of horse racing. However, it can also be a great guide for those who bet on horse racing. The news pieces show amazing insight into the world of horse racing and could help bettors improve their odds of winning.

Want a PROVEN system to make money gambling? Click Here to find out more!

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How to travel within your budget

Are you wondering can anyone really travel within their budget? With the way how people end up today with no jobs, and no steady reliable income source flowing in, for such people going on vacation is considered to be lucky. Also you will want to keep a budget for the time spent away from home. For people who have limited budget, planning a vacation is also possible, just follow the given tips and enjoy your trip.

First, you?re going to need to pay for your airfare and hotel accommodations. You can go online and find some cheap prices for both airfare and hotel accommodations in a package deal, and if you are lucky you can also find cheaper prices on the touring facility for tourist spots in the surrounding the area that you will be staying in. Do some research and make sure to get the lower prices.

Once you pay for your airfare and hotel accommodations you?ll want to figure out how much you can spend each day on your vacation. Figure out the amount of money you have and things you would want to buy or other ticket fares to the tourist places. If you have few thousands to spend on a vacation and you?ll be there for one or two weeks, you?ll have the luxury of spending couple of hundreds each day and try to keep all your expenses under that amount. You can keep a log of what you are spending each day so that you will know exactly what is left and how much you have spent. If you don?t spend that much in one day, carry it over to the next day.

If you think there is absolutely no other way you can go on a vacation, ask for a payday loan and go traveling! To stay within your budget, this is another way you can do it without sacrificing your self pleasure. If your company gives out loans then you can make it work for you and your family. Payday loans do come with interest though and if you feel you can repay a payday loan on time, then give it a shot and indulge yourself and your family in a fun-filled vacation.

If you really want to keep within your budget, you might want to take day trips around the city or town you are in. That can be fairly cheap and may even work out better with you budgeting so that you can eliminate the airfare and the hotel too.

You can also try camping. It?s fun and it?s inexpensive. You can go with your friends and have a blast, if you fish or like to hike, this can be a good vacation for you too! You have so many options to choose from that can and will save you money.

Neo Zack is a Tech writer from London with an interest in topics relating to Travel, Insurance, Finance, and green living. You can follow him @financeport on Twitter.

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Tips to Help You Add Quickly

Features | More Science

The Math Dude: Quick and Dirty Tips to Make Math Simpler

By Jason Marshall ?| June 27, 2012

Scientific American presents Math Dude by Quick & Dirty Tips. Scientific American and Quick & Dirty Tips are both Macmillan companies.

Today we're turning our attention to solving math problems faster than you ever thought was possible. In this article you?ll learn two tips to help you add quickly?all in your head! Next we'll build on these tips to learn another way to do some fast addition in your head.

Tip #1: Find Pairs of Numbers that Add to 10
Here is perhaps the single most useful quick and dirty tip to help you calculate quickly: When adding a list of numbers, look for pairs of numbers that add to ten.

> Continue reading on QuickAndDirtyTips.com

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Focus on the Family Community: Finding Home: When Your Pet Dies

There were two dogs throughout my youth, a pug named Sir George and a Great Dane named Duke.

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I guess the Dalys felt a certain kinship to English royalty, even as I poured Kool-Aid over my Cheerios. (There wasn?t always enough money for milk.)

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Duke and Sir George were my sister?s dogs and so in many ways, I had the best of both worlds. I? enjoyed playing with them under the warm California sun, but never had to feed or tend to their needs. She took care of the essentials. I liked that. When they died, I cried, but I was still young and had already dealt with so much dysfunction that I don?t remember their deaths striking me too hard.

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I grew up, married Jean, and soon found myself with a cat, which didn?t necessarily appeal to me, because I?ve always considered myself a dog person.

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But Tommy didn?t act like a cat. He thought he was a dog!? He would greet me by running in circles and dashing through my legs.? His end came suddenly, at the ripe old age of fifteen. He had fallen on a nail and succumbed to a fast-moving blood disease.

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I didn?t think his death would bother me, but it struck me fairly hard. This was in the days before children, so I hadn?t even realized how attached I had become to him.

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This is the curious and peculiar thing about our emotional pull toward our pets.

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They don?t talk. They take.

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Yet maybe it?s because they don?t talk back and they express unfailing loyalty that we grow so close to them so quickly. They become a part of the family and as such, when they die, sometimes we feel like a part of us dies, too.

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This is probably what Steve Allen meant when he once said that ?Old men miss many dogs.? I?m sure many of you can relate.

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The late southern writer, Willie Morris, whose sentimental book, My Dog Skip, was turned into a heartwarming film in the 1990s, may have said it best:

The dog of your boyhood teaches you a great deal about friendship, and love, and death: Old Skip was my brother. They had buried him under our elm tree, they said?yet this wasn't totally true. For he really lay buried in my heart.

Because God is the creator of every living thing, and the giver of every good gift, we have Him, of course, to thank for our pets, even a cat named Tommy who thought he was a dog.

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I'm curious. Do you have a story to share about a special pet in your life?

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Control4 delivers home automation Starter Kit for under $1,000 including installation, we go hands-on

Replacing a house full of switches and dials with a single remote seems a desirable proposition, but less-than-intuitive setup processes and fees that run far into the thousands make a disconnected home the only option for some. Like other home automation system manufacturers, Control4 has a bounty of offerings that can run up pricing into that out-of-reach range, but if you're looking to adjust audio and video in a single room, the new Starter Kit should get you going for just shy of a grand, including installation. That price includes a HC-250 Controller, which delivers IR control for up to eight components (via splitters connected to the four IR ports on the rear), serial control for up to two receivers or other systems and IP control for an unlimited number of devices. There's also an SR-250 ZigBee remote in the box, which offers full control through a television interface (HDMI and component outputs can be found on the HC-250's rear). You can also have full access through a variety of add-ons, including a $999 7-inch in-wall touchscreen with camera, a portable version for the same price, or any Android, iOS or Mac device -- access licenses for smartphones, tablets and computers run $199 each, or $499 to cover the entire home.

The Starter Kit can enable control of a single room, which may be fine for some users -- to add additional home theater setups you can bring on more HC-250s at $599 a pop. There's also an option to add ZigBee lighting controls ($129 per switch), ZigBee door locks ($150 to $350) or a door intercom unit with camera ($799). All-in, outfitting a large home can be quite pricey, and the Starter Kit is designed to get folks in the door, rather than to deliver a complete solution. We tested the controller with a TV, audio system, a pair of lights on two zones, the door intercom and a deadbolt, and all performed seamlessly without an hiccups. We also took a look at the intuitive drag-and-drop PC-based interface, which owners can use to change macros and add media. Introducing new components to the rig will require a dealer service call (or remote access, if you're just trying to loop in something like a NAS to serve up content). The Control4 Starter Kit is available through third-party dealers beginning today, including Magnolia Home Theater in select Best Buy stores (in that case, Geek Squad will handle the install). That sub-$1,000 figure factors in two hours of labor, and may climb a bit higher depending on dealer rates. Still, if you're just looking to get your feet wet, this seems to be a solid solution. Thumb through the gallery below for a closer look at the components and interface.

Gallery: Control4 Starter Kit hands-on

Continue reading Control4 delivers home automation Starter Kit for under $1,000 including installation, we go hands-on

Control4 delivers home automation Starter Kit for under $1,000 including installation, we go hands-on originally appeared on Engadget on Thu, 28 Jun 2012 11:33:00 EDT. Please see our terms for use of feeds.

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SME Webinar: How to Tackle the Biggest ... - Social Media Explorer

We know online retailers are battling with real-time consumer demands, negative online reviews and shopping cart abandonment from busy consumers. Plus, we add in the demands of integrating social media and plates start overflowing. The challenges are real and the solutions aren?t always easy. So the Social Media Explorer team is happy to bring you a useful and informative webinar to give you insights right from some of the industry?s leading experts.

Hear how expert online retailers are dealing with:

• Tips to decrease shopping cart abandonment
• How to integrate social media into the online shopping experience
• Ways to use social sharing to drive new business
• Tips to balance the needs of real-time consumer demands and business objectives
• How to adjust to consumer demands for online purchase behaviors
• Ways to maximize customer service experiences

Register Now

Wednesday, July 11, 2012
1 p.m. ET/10 a.m. PT

Our panel of experts offer decades of experience in handling all of these issues and have successfully turned challenges like these into opportunities to grow their online retail businesses driving millions in revenue. Jason Falls will host a fireside chat with?Barry Litwin, former VP of Global Commerce for Office Depot; Brad Butler, COO of Halloween Express and Jeff Mason, VP of Marketing for Velaro to get the inside scoop.

1. Hear how Barry Litwin grew the online retail business for Office Depot to a $4.1 million web business that was ranked #5 by Internet Retailer.
2. Imagine having to do 90% of your revenues in less than 2 months! Brad Butler will explain how Halloween Express drives sales in a highly seasonal business without adding a second Halloween to the calendar!
3. Want a little variety in your online retail diet? Jeff Mason has worked with hundreds of online retailers to implement chat solutions and can tell you exactly what the leaders are doing right and what the losers are doing wrong.

Barry Litwin ? Retail E-Commerce Executive and Thought Leader

Barry is a frequent internet industry speaker, and a seasoned e-commerce and mobile executive having led e-commerce for several large scale retail and multi-channel companies. He was VP, Global E-Commerce for Office Depot?s $4.1M web business, ranked #5 by Internet Retailer, where he oversaw strategic planning, on-line merchandising, functional innovation, site usability and design, and web analytics. He was also responsible for mobile strategy and omni-channel experience initiatives. Previously, he was with $1.2B Premier Farnell, as the SVP, E-Commerce and Marketing for the North American Newark Electronics brand. He led digital marketing, e-commerce development, and multi-channel marketing. Barry was general manager for Block & Company, Inc., a $100M multi-channel distributor of money handling products to financial and gaming customers, and has also held senior leadership roles in sales and direct marketing at Office Max and Archibald Candy Company.

Brad Butler Jr. ? Internet Entrepreneur, Chief Operating Officer ASADART E-Commerce Specialty Shops

Since 2005 Brad has been the driving force behind the online presence of one of the country?s largest Halloween retailers, Halloween Express. Under Brad?s leadership HalloweenExpress.com has established itself as a dominant online player in the highly competitive Halloween seasonal business. Brad is directly responsible for the day-to-day management of the company?s ecommerce operations which among other things includes direct control over all marketing, SEO and social media marketing efforts. Additionally, Brad is the managing partner of Habco Services Group which provides online marketing consulting services to select companies. Prior to jumping into the online marketing arena, Brad was the founder, President and CEO of one of the largest telecommunications service bureaus in the country with nearly 500 employees.

Jeff Mason ? Technology Entrepreneur, Vice President of Marketing for Velaro, Inc.

With more than?20 years of technology marketing experience,?Jeff Mason is the Vice President of Marketing for Velaro,?a leading provider of live chat software. He and his team have worked with countless online retailers to improve customer service, decrease shopping cart abandon rates, and drive more revenues with the implementation of their software. Velaro believes live chat software is the solution for extending social conversations to a corporate website. Jeff was also Vice President of Marketing for Social Solutions, he co-founded Artifact Software, and was ?a member of Sequoia Software?s executive management team. When Sequoia was acquired by Citrix, Jeff served on the company?s management team and established Citrix Solutions Marketing Group. Throughout his career, Jeff has shown a passion for leveraging technology to improve customer experiences while driving revenue. He provides a unique perspective on where online retailers are missing the opportunity to engage their website visitors.

Register Now

Wednesday, July 11, 2012
1 p.m. ET/10 a.m. PT

And if you have questions you?d like to ask ahead of time, dive into the comments and file them. We?ll review before the webinar and try to get them answered!

The Comments are yours!

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New planet-weighing technique found

ScienceDaily (June 27, 2012) ? Although there have been about 800 extra-solar planets discovered so far in our galaxy, the precise masses of the majority of them are still unknown, as the most-common planet-finding technique provides only a general idea of an object's mass. Previously, the only way to determine a planet's exact mass was if it transits -- has an orbit that periodically eclipses that of its host star. Former Carnegie scientist Mercedes L?pez-Morales has, for the first time, determined the mass of a non-transiting planet.

The work will be published in Astrophysical Journal Letters.

Knowing a body's mass is essential first to confirm it is a planet and if so, to determine whether it is rocky and possibly habitable or large and gassy. Until now, only the masses of transiting planets have been measured. Transiting planets are also the only type of extra-solar objects on which atmospheres have been detected.

L?pez-Morales, along with her colleagues Florian Rodler and Ignasi Ribas of the Institute of Space Sciences, ICE (CSIC-IEEC, in Barcelona, Spain) measured the exact mass of a non-transiting planet. They did this using a new method that involves studying the carbon monoxide signature of the planet's atmosphere -- detecting, in the process, the atmosphere of this non-transiting planet.

The planet is called Tau Boo b, located in the constellation of Bootes, and it orbits a star about 50 light years from Earth that's bright enough to be visible to the naked eye. The planet is similar in size to Jupiter and is so close to its star (only 8 stellar radii), that a year for this planet asts only 3.3 Earth days. Furthermore, its surface temperature reaches 1,500 ? C, making it inhospitable to life.

Discovered in 1996, Tau Boo b was one of the first planets originally detected by the radial velocity method. This planet does not transit, but its presence and characteristics were initially determined by the wobble of its host star. This technique only provides a rough indication of a detected planet's mass.

In June 2011, L?pez-Morales' team conducted five hours of observations at near infrared wavelength (2.3 microns). They obtained data from the high-resolution spectrograph CRIRES, an instrument mounted on one of the four 8.2m Very Large Telescopes (VLT) of the European Southern Observatory (ESO) in Chile.

The observations and subsequent data analysis revealed the presence of carbon monoxide in the planet's atmosphere. In addition, by studying the planet's orbital motion through the displacement of spectral lines of carbon monoxide, the team was able to calculate its exact mass -- 5.6 times Jupiter -- a first using this particular method, and also a first for a non-transiting planet.

An independent study conducted by researchers at the University of Leiden in the Netherlands obtained a similar result for the same planetary system, confirming the potential of this technique.

"This method represents a strong advance in the field of exoplanets," said Lopez-Morales. "It opens a new path to determine masses of exoplanets and the composition of their atmospheres"

The research team expects many more planets will be weighted using this new technique. They are also convinced that in the future, they will be able to detect molecules that are associated with the presence of life in non-transiting distant planets."

This work has been partially supported by the NSF.

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Google+ Wants You to Fill It With Updates From Other Sites [Google+]

Venture Beat is reporting that there's an extra Google+ feature that wasn't announced at yesterday's I/O, which sucks content from other sites for you. Sounds like it might be only way you'll ever post anything to your profile. More »

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Ways To Improve Your Wellness With Physical fitness | ApostolicCM ...

You need to take care of your yourself, no matter exactly how old you are. There are numerous tell-tale indications that indicate your total degree of health. Your fitness degree is incredibly essential to your wellness. Right here are some suggestions to help you establish an excellent physical fitness program.

Beginning an extensive new workout session program can easily be remarkably overwhelming, specifically if you organize to work with a fitness instructor. If you are fretted that you might not follow through with your commitment, pay your trainer the complete quantity up front. You will definitely be less most likely to skip workout sessions if you have currently made a substantial financial investment.

Spinning classes can be a wonderful entertainment way to get in shape. Many individuals go to the gym deciding that they entirely prefer to concentrate on aerobic workout to shed pounds. Well, spinning is just one of the greatest means to reduce weight due to the fact that it takes out the absolute quantity of stress on your joints that you may receive from long distance running, while still pairing it with a calorie-burning aerobic task.

A wonderful physical fitness tip is to ensure you concentrate on lifting weights with great form. A ton of rookie weight lifters get held away with lifting hefty weights and they wind up dropping their form. This can easily get you seriously injured. Lifting with great type is vital.

Summer time heat can really make it challenging to get out and get the exercise that you want and need to get. Try to drink a low calorie slushie prior to or after your run. It will certainly cool your body temperature down and provide you a rejuvenating way to rehydrate after a long run.

Do not think of the procedure of getting fit as a short-term exercise with a cut-off date. Health and fitness is a lasting commitment. In fact, it should be a permanent one. When constructing a physical fitness regular think about not just the instant perks however whether the program is one that can be sustained indefinitely. Physical fitness is for life, not merely for swimsuit season.

If you are able to, workout 1st thing in the morning. It will definitely get your metabolism selecting the day. Feeling slow mid-morning or mid-afternoon? Get up from whatever you?re doing and take a 10-15 minute walk. Beverage a couple of bottles of water while you?re at it. Do not get that candy bar !!!

When you drop many inches on your waistline, attempt new clothing! You can observe how much weight you?ve lost by trying on brand-new pairs of pants and clothing that you formerly may have never thought you could. Using those clothing you never thought were feasible can be a wonderful inspirational booster to individuals that would like to gain a far better figure.

Locate means to make exercising enjoyable. Activity is very important, however it can be hard to continue if it feels like a task. Vary your regular by running or walking a different course, joining a sports team, or making use of tasks like gardening or dancing to exercise. Making activity fascinating will definitely make it a satisfying routine.

While tackling health and fitness dealing straight with your biceps, there are numerous exercises that will especially assist this location. Focused bicep curls, hammer curls, and the preacher curl all work effectively in working out your biceps. These are exceptional workouts to get you on the pathway of much better workout. Remember, biceps are the 1st muscle individuals examine to judge your strength.

If you have a desk job and are worried about staying fit, consider keeping a mini-stepper under your desk and utilize it for a few moments of every hour. Also 5 moments of physical fitness per hour will definitely make a large difference. This will definitely also help prevent the soreness and rigidity connected with prolonged periods of inactivity.

In conclusion, health and fitness is incredibly vital to your overall well being. There are great deals of noticeable physical fitness related things that you can easily do in addition to things that you could never ever have actually thought about. As long as you follow the strategies and tricks in this write-up you ought to locate much success.

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