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Experimental Therapeutics, Molecular Targets and Chemical Biology
Cyclooxygenase-2 Inhibits Novel Ginseng Metabolite-Mediated Apoptosis
Hyung Woo Yim, Hyun-Soon Jong, Tai Young Kim, Hyun Ho Choi, Sang Gyun Kim, Sang Hyun Song, Juyong Kim, Seong-Gyu Ko, Jung Weon Lee, Tae-You Kim and Yung-Jue Bang
Hyung Woo Yim
Hyun-Soon Jong
Tai Young Kim
Hyun Ho Choi
Sang Gyun Kim
Sang Hyun Song
Juyong Kim
Seong-Gyu Ko
Jung Weon Lee
Tae-You Kim
Yung-Jue Bang
DOI: 10.1158/0008-5472.CAN-04-1740 Published March 2005
Recently, a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, i.e., 20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), has been reported to induce apoptosis in a variety of cancer cells. Here we show a differential effect of IH-901 on several cell types. Exposure to IH-901 for 48 hours at a supposedly subapoptotic concentration of 40 μmol/L led to both apoptotic cell death and G1 arrest in Hep3B cells, but only resulted in G1 arrest in MDA-MB-231, Hs578T, and MKN28 cells. Additionally, the treatment of MDA-MB-231, but not of Hep3B, with IH-901 up-regulated cyclooxygenase-2 (COX-2) mRNA (2 hours) and protein (6 hours), and enhanced the production of prostaglandin E2. In MDA-MB-231 cells, IH-901 induced the sustained activation of extracellular signal-regulated kinase (ERK), whereas inhibition of mitogen-activated protein/ERK kinase blocked IH-901-mediated COX-2 induction and resulted in apoptosis, suggesting the involvement of an ERK-COX-2 pathway. Combined treatment with IH-901 and nonsteroidal anti-inflammatory drugs inhibited COX-2 enzyme and induced apoptosis in MDA-MB-231 and Hs578T cells. Adenovirus-mediated COX-2 small interfering RNAs also effectively inhibited COX-2 protein expression and enhanced IH-901-mediated apoptosis without inhibiting ERK 1/2 phosphorylation, thus providing direct evidence that COX-2 is an antiapoptotic molecule. Moreover, IH-901-mediated G1 arrest resulted from an increase in p27Kip1 mRNA and protein expression followed by a decrease in CDK2 kinase activity that was concurrent with the hypophosphorylation of Rb and p130. In conclusion, IH-901 induced both G1 arrest and apoptosis, and this apoptosis could be inhibited by COX-2 induction.
IH-901
COX-2
p27Kip1
Panax ginseng, C.A. Meyer, is an herbal root that has been used for more than 2000 years throughout Far Eastern countries including China, Japan, and Korea. Its beneficial effects have been analyzed by extensive preclinical and epidemiological studies (1–3) . Recently, 20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel ginseng saponin metabolite, formed from ginsenosides Rb1, Rb2, and Rc was isolated and purified after giving ginseng extract p.o. to humans and rats (4). IH-901 has been shown to enhance the efficacy of anticancer drugs in cancer cell lines previously resistant to several anticancer drugs (5, 6) , to exhibit antigenotoxic and anticlastogenic activity in rats concurrently treated with benzo(a)pyrene (7), and to induce apoptosis (8, 9) . These studies found that the antitumor activity of IH-901 is attributable to the induction of apoptosis.
Recently, the link between cyclooxygenase (COX)-2 and cancer has been studied (10, 11) . COX catalyzes the conversion of arachidonic acid to prostaglandins and related eicosanoids. Two isoforms of COX, which are the products of distinct genes, have been identified. COX-2 is an inducible and immediate-early gene that is up-regulated in many epithelial malignancies (12–15) . Unlike constitutively expressed COX-1, COX-2 overexpression is associated with neoplastic transformation (12), cell growth (16), angiogenesis (17), invasiveness, and metastasis (18). Although in vivo, the exact mechanism has not been clearly elucidated, in vitro studies have shown in a variety of cell types that COX-2 overexpression is antiapoptotic (19). This antiapoptotic effect is associated with inhibition of Fas (20) and up-regulation of Bcl-2 (21). Even though COX-2 is an important target for cancer therapy, COX-2 is induced by several anticancer agents such as paclitaxel (22), mitomycin C (23), and radiation (24), suggesting its association with drug resistance.
Physiologically, p27Kip1 seems to be important for blocking progression of cells from late G1 into S via its interaction with cyclin E-CDK2 complexes (25). p27Kip1 has been also implicated as a mediator of the growth arrest due to its regulation by protein kinase A activating agents (26). IH-901 stimulated p27Kip1 induction and subsequent cell cycle (G1) arrest.
In this study, we discovered that IH-901, a novel ginseng metabolite, could inhibit human cancer cell proliferation by inducing cell cycle arrest as well as apoptosis. Treatment with IH-901 resulted in cell cycle (G1) arrest by p27Kip1 induction. Additionally, we provide evidence here that COX-2 functions as an antiapoptotic molecule as its inhibition by both nonsteroidal anti-inflammatory drugs (NSAID) and adenoviral-COX-2 small interfering RNAs (siRNA) significantly enhanced IH-901-mediated apoptosis. These findings provide a molecular basis for the antineoplastic effects of IH-901 and use of this ginseng metabolite may aid in enhancing the anticancer drug efficacy by COX-2 inhibition.
Reagents. IH-901 was kindly provided by Dr. J-H. Sung (Central Research Institute, Il Hwa Co., Ltd., Korea) and was previously described (9). NS-398 was purchased from Cayman Chemicals (Ann Arbor, MI); actinomycin D from Sigma Chemical Co. (St. Louis, MO); CDK2 (M2), cyclin E (C-19), Bax and COX-2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); p21WAF1/CIP1 (C24420) and p27Kip1 (K25020) antibodies from Transduction Laboratories (Lexington, KY); pRB (G3-245) antibodies from PharMingen (San Diego, CA); and Bcl-2 antibodies from DAKO (Denmark).
Cell Cultures. MDA-MB-231, Hep3B, and MKN28 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and gentamicin (10 μg/mL). Hs578T cell line was grown in DMEM supplemented with 10% fetal bovine serum, and gentamicin. Subconfluent monolayers of cells were utilized in all experiments.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazoliumbromide Assay. Cell viability was determined by measuring the absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (Sigma) dye for living cells, as described earlier (9). IC50's were calculated based on the survival rate of untreated cells being 100%.
Cell Cycle Analysis. Apoptosis was monitored by measuring hypodiploid DNA content (sub-G1). Cells (5 × 105) were seeded in 60 mm dishes and allowed to attach overnight. Cell cultures were then incubated with IH-901 at 40 μmol/L concentration for the indicated times. fluorescence-activated cell sorting analysis using propidium iodide (PI) was done as described previously (27). The relative DNA content per cell was obtained by measuring the fluorescence of DNA bound propidium iodide. Cell cycle analysis was conducted using a FACStar flow cytometer (Becton Dickinson, San Jose, CA) and a ModFit LT V2.0 computer program.
Chromosomal DNA Isolation and Ladder Formation Assay. Cells were harvested and then suspended in a lysis buffer [50 mmol Tris-Cl (pH 8.0), 10 mmol EDTA, and 0.5% laurylsarcosine] containing 50 μg/mL RNase A. Chromosomal DNA was isolated and ladder formation assays were done as described previously (9).
RNA Extraction, Northern Blot Analysis, and mRNA Stability Assay. Total RNA preparation and Northern blotting were done as described previously (28). Blots were hybridized with radiolabeled probes for COX-2 cDNA (kindly provided by Dr. Stephan M. Prescott, University of Utah) and a p27Kip1 cDNA fragment for RNA expression. For mRNA stability assay, the relative band intensities of p27Kip1 and β-actin mRNA at each time point were quantified by determining radioactivity using a Fuji FLA2000 and image analysis software (Image Gauge, version 3.12).
Western Blot Analysis. Cells were washed with ice-cold PBS and suspended in lysis buffer [20 mmol Tris-Cl (pH 7.4), 100 mmol NaCl, 1% NP40, 0.5% sodium deoxycholate, 5 mmol MgCl2, 0.1 mmol phenylmethylsulfonyl fluoride, 0.1 mmol pepstatin A, 0.1 mmol antipain, 0.1 mmol chymostatin, 0.2 mmol leupeptin, 10 μg/mL aprotinin, 0.5 mg/mL soybean trypsin inhibitor, and 1 mmol benzamidine] on ice for 30 minutes. Lysates were cleared by centrifugation at 13,000 rpm for 20 minutes. Equal amounts of cell extracts (100 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with appropriate primary and horseradish-conjugated secondary antibodies, as described previously (29). Anti-α-tubulin antibody (Sigma) was used as a loading control. Detection was done using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).
Prostaglandin E2 Production and Determination of COX-2 Activity. Cells were treated with IH-901 and/or celecoxib, a specific COX-2 inhibitor, for 24 hours in complete growth medium containing 10% fetal bovine serum. Afterwards, culture medium was collected to determine the amount of prostaglandin E2 (PGE2) secreted by the cells. For the measurement of COX-2 enzymatic activity, PGE2 quantification was measured using a PGE2 enzyme immunoassay kit (Cayman) according to the manufacturer's instructions, as described previously (30). Results are expressed as picograms of PGE2 per 106 cell ± SD.
Immunoprecipitation and Kinase Assays. CDK2 and cyclin E-associated H1 histone kinase activity were determined as described previously (27). Two hundred micrograms of cell extracts were used per immunoprecipitation with CDK2 antibody coupled to protein A or G beads. After being washed, CDK2 kinase assays on histone H1 was done by incubating the immune complex beads with 30 μL of kinase reaction [0.25 μL (2.5 μg) of histone H1, 0.5 μL (5 μCi) of γ-[32P]ATP, 0.5 μL of 0.1 mmol ATP and 28.75 μL of kinase buffer] for 30 minutes at 37°C. The reaction was stopped by boiling the samples in 2× SDS sample buffer for 5 minutes. Samples were analyzed by 12% SDS-PAGE, and the gels were then dried and subjected to autoradiography. Preimmune serum was also analyzed for possible nonspecific immunoprecipitation in these studies.
Construction of Recombinant Adenoviral Vector of siRNAs for COX-2 Gene. pSUPER vector (31) enabled us to directly clone synthetic oligonucleotides yielding U1 promoter–based knock-down constructs. Target sequences of the siRNA specific for COX-2 and GFP duplex are as follows: siRNA-COX-2#1 (M90100), 5′-AAC CGA GGT GTA TGT ATG AGT GT-3′; siRNA-COX-2#2, 5′-AAT GCA ATT ATG AGT TAT GTG TT-3′; and siRNA-pEGFP (U57609), 5′-GGC TAC GTC CAG GAG CGC ACC-3′. Both siRNAs against COX-2 were controlled for sequence specificity by BLAST searching and did not show homology to other known human genes, especially COX-1. SpeI-EcoRI digested pSUPER containing U1 promoter–directed knockdown constructs were subcloned into pShuttle vector of Adeno-X Expression System (Clontech, Palo Alto, CA), thereby replacing the cytomegalovirus-promoter sequence. The sequences of the inserts and their orientations were examined by DNA sequencing analyses. siRNA expression cassettes for COX-2 or GFP from recombinant pShuttle plasmid DNA were digested and ligated to Adeno-X viral DNA according to the manufacturer's instructions. These adenoviral vectors were infected and amplified in HEK293 cells. CsCl-purified virus was dialyzed against Ca2+- and Mg2+-free DPBS containing 10% glycerol. The virus titer was determined using a standard plaque assay. Viral stock was aliquoted into appropriate volumes and stored at −70°C until use.
Adenovirus Infection In vitro. Recombinant adenoviruses expressing COX-2 or GFP siRNAs were used on the target cells at a multiplicity of infection of 20, and incubated at 37°C for 2 hours in a CO2 incubator. The medium was removed and cells were then cultured for 24 hours before further experimentation.
Data Analysis. Results are representative of at least three independent experiments done in triplicate and are presented as mean ± SD. Comparisons between groups were analyzed using Student's paired t test.
Effect of IH-901 on Cell Proliferation of Cancer Cells. We examined the effect of IH-901 on cellular proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. Significant dose-dependent growth inhibition was observed in two breast cancer cell lines (MDA-MB-231 and Hs578T), as well as gastric (MKN28), and hepatoma (Hep3B) cell lines following treatment with IH-901 for 3 days ( Fig. 1 ). The concentration required for 50% inhibition of growth (IC50) by IH-901 ranged from 33.1 to 42.5 μmol/L. These results are consistent with earlier reports using myeloma cells (8, 9) . In order to examine the mechanism of growth suppression by IH-901, cancer cells were treated with 40 μmol/L IH-901 for 48 hours and then analyzed for cell cycle progression by flow cytometry. The apoptotic sub-G1 fraction increased to 31.74% in Hep3B cells after treatment with IH-901 for 48 hours, whereas those of MDA-MB-231 and MKN28 cells were 5.23% and 2.76%, respectively, indicating that IH-901 induced a significant apoptotic cell death in Hep3B but not in MDA-MB-231 and MKN28 cells ( Fig. 2A ). The increase in the sub-G1 fraction and endonucleolytic DNA cleavage in Hep3B cells by IH-901 was evident in a time-dependent manner ( Fig. 2B). As shown in Fig. 2C, IH-901-induced cell cycle arrest at the G0-G1 phase and a progressive decline of S phase cells occurred in MDA-MB-231 as well as in Hep3B cells. These results suggest that IH-901 induced apoptotic cell death concurrent with cell cycle arrest in Hep3B cells, whereas it caused G0-G1 arrest only in MDA-MB-231 and MKN28 cells. As IH-901-mediated apoptosis is reported to be associated with the activation of caspase-3 and PARP cleavage (8, 9) , levels of antiapoptotic Bcl-2 and proapoptotic Bax proteins were analyzed in Hep3B cells. Bcl-2 expression decreased in a time-dependent manner but Bax expression did not change ( Fig. 2D). Hs578T breast cancer cell lines also showed only G0-G1 arrest after treatment with 40 μmol/L IH-901 (data not shown).
Growth inhibitory effects of IH-901 on MDA-MB-231 and Hs578T breast cancer cells, MKN28 gastric cancer cells, and Hep3B hepatocellular carcinoma cells. Exponentially growing cells were treated with the indicated concentration of IH-901 for 72 hours. Cell growth inhibition was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, as described in Materials and Methods. Inset, IC50 values of the individual cell lines. The assay was done using six replicates and repeated more than thrice. Points, mean; bars, ± SE.
Cell cycle analysis of MDA-MB-231, MKN28, and Hep3B treated with 40 μmol/L IH-901 at the indicated times. After cells had been fixed and stained propidium iodide, the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using a FACStar flow cytometer (Becton Dickinson) A, percentages of sub-G1 phase cells which were determined based on DNA content histogram (inset). B, induction of chromosomal DNA fragmentation by IH-901 in Hep3B cells. Chromosomal DNA was extracted from cells treated with IH-901 for the indicated times, and electrophoresed in a 1.5% agarose gel, as described in Materials and Methods. The DNA size markers, λ/HindIII and ΦX174/HaeIII were used as controls. C, the population percentages of G1 and S phase from each cell cycle phase were determined based on DNA content in (A) with viable nonapoptotic cells (≥2 N DNA contents). D, the levels of antiapoptotic Bcl-2 and proapoptotic Bax proteins were analyzed in Hep3B cells after the treatment with IH-901 for the indicated times.
COX-2 Induction by Treatment with IH-901. Because several recent reports have implicated the role of COX-2 in preventing apoptosis in various cell types (19–23) , we examined the ability of IH-901 to induce COX-2 in MDA-MB-231 cells. As shown in Fig. 3A , 40 μmol/L of IH-901 treatment resulted in the induction of COX-2 protein in MDA-MB-231 cells. COX-2 protein and mRNA were induced in a time-dependent manner by 40 μmol/L IH-901; the ethanol vehicle had no effect on COX-2 induction ( Fig. 3B). PGE2 production was also increased by IH-901 treatment in a dose-dependent manner, consistent with the induction of COX-2 protein ( Fig. 3C). IH-901 also induced COX-2 protein in MKN28 and Hs578T cells but did not lead to COX-2 induction in Hep3B cells ( Fig. 3D). Mitogen-activated protein kinase (MAPK) pathways mediate the regulation of COX-2 expression when induced by a variety of extracellular stimuli (32, 33) . To determine whether IH-901 activates MAPK pathways in MDA-MB-231 cells, the levels of phosphorylated extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun-NH2-kinase proteins were evaluated by Western analysis. Western blots of MDA-MB-231 cell extracts were probed with antibodies selective for the phosphorylated (activated) forms of ERK-1 and ERK-2. As shown in Fig. 4A , IH-901 induced a time-dependent increase in phosphorylated ERK-1 and ERK-2 with levels elevated up to 24 hours. Marginal ethanol-induced ERK activation was also observed but this was very weak compared with IH-901-induced ERK activation (data not shown). To determine whether the activation of ERK-1 and ERK-2 is required for IH-901-mediated COX-2 induction, MDA-MB-231 cells were pretreated with UO126, a selective inhibitor of MAP/ERK kinase 1/2. UO126 (10 μmol/L) strongly inhibited IH-901-induced COX-2 expression for up to 12 hours (> Fig. 4A). Although blocking ERK1/2 with 10 μmol/L UO126, the cell cycle distribution of MDA-MB-231 cells was analyzed by flow cytometry ( Fig. 4B). Cotreatment with IH-901 and UO126 caused the percentage of apoptotic sub-G1 population to increase from 5% to 18%, compared with IH-901 treatment alone, suggesting that MAP/ERK kinase inhibition can enhance IH-901-mediated apoptosis in MAD-MB-231 cells. No significant change in the phosphorylated forms of c-jun-NH2-kinase and p38MAPK was observed until 4 hours of IH-901 treatment ( Fig. 4C).
IH-901 induced COX-2 in MDA-MB-231, MKN28, and Hs578T cells but not in Hep3B cells. A, MDA-MB-231 cells were harvested after being incubated with the indicated concentrations of IH-901 for 24 hours. Equal amounts of cell extracts (100 μg/lane) were resolved by 10% SDS-PAGE and analyzed by Western blotting with an antibody specific for COX-2. B, treatment of MDA-MB-231 cells with 40 μmol/L IH-901 induced COX-2 expression of mRNA and protein in a time-dependent manner but vehicle (ethanol) did not. Fifteen micrograms of total RNA were used for Northern analysis. Anti-α-tubulin was used as a protein loading control. C, MDA-MB-231 cells were treated with varying concentrations of IH-901 and/or celecoxib for 24 hours. Conditioned media were collected and analyzed by ELISA for PGE2. Results are expressed as the mean of six separate experiments done in duplicate. Columns, mean; bars, SD; n = 6. *, P < 0.01 compared with untreated cells. IH-901 increased PGE2 production in a dose-dependent manner, and celecoxib effectively reduced IH-901-mediated PGE2 production. D, MDA-MB-231, Hs578T, MKN28, and Hep3B cells were treated with 40 μmol/L IH-901 for 24 hours. Western analyses were done with antibodies specific to COX-2 or α-tubulin.
IH-901 induced a time-dependent increase in phosphorylated ERK1/2 in MDA-MB-231 cells. Equal amounts of cell extracts (100 μg/lane) were resolved by 10% SDS-PAGE and analyzed by Western analysis with antibodies specific to ERK1/2 or to the phosphorylated forms of ERK1/2. COX-2 induction was significantly inhibited by MAP/ERK kinase 1/2 inhibitor (UO126) in MDA-MB-231 cells. A, to determine whether MAPK activation is required for IH-901-induced increases in COX-2 protein levels, cells were treated with a selective MAP/ERK kinase inhibitor (UO126) 1 hour before 40 μmol/L IH-901 or vehicle addition and then incubated for 24 hours. UO126 (10 μmol/L) significantly inhibited COX-2 induction by IH-901. B, MDA-MB-231 cells were preincubated with 10 μmol/L UO126 for 60 minutes. Afterwards, the addition of IH-901 increased the apoptotic sub-G1 fraction, whereas UO126 treatment alone showed no toxic effect on MDA-MB-231 cells. The percentage of sub-G1 cells was determined based on their DNA content histograms. Representative data from three independent experiments. C, MDA-MB-231 cells were treated with ethanol vehicle and IH-901 for the indicated times and Western analyses of the activated form and total p38 and c-jun-NH2-kinase expression were done. Phorbol 12-myristate 13-acetate was used for the induction of c-jun-NH2-kinase activation.
Inhibition of COX-2 Enhances IH-901-Mediated Apoptosis. Celecoxib inhibited IH-901-induced production of PGE2 in a dose-dependent way in MDA-MB-231 cells ( Fig. 3C). To address whether COX-2 protein induced by IH-901 plays an important role in protecting cells from IH-901-mediated apoptosis, we examined the effects of COX-2 inhibition on IH-901-mediated apoptosis using the selective COX-2 inhibitors, celecoxib, or NS-398 ( Fig. 5A ). Briefly, MDA-MB-231 cells were treated with celecoxib (25 or 100 μmol/L) or NS-398 (100 or 500 μmol/L) for 30 minutes before adding 40 μmol/L IH-901. Cell cycle analysis showed that although celecoxib and NS-398 alone had no significant effect on the apoptotic fraction, pretreatment with celecoxib (25 μmol/L) followed by IH-901 enhanced the apoptotic sub-G1 fraction from 5% to 40%. Similarly, NS-398 (500 μmol/L) pretreatment followed by addition of IH-901 increased the apoptotic fraction up to 30%. The combined treatment of a COX-2 inhibitor (25 μmol/L celecoxib or 200 μmol/L NS398) with 40 μmol/L IH-901 increased the apoptotic sub-G1 fraction in Hs578T cells. These results strongly suggest that COX-2 enzyme activity protects MDA-MB-231 and Hs578T cells from IH-901-induced apoptosis. We analyzed the expression of Bcl-2 and Bax in the absence or presence of NS-398 (250 μmol/L) on 40 μmol/L IH-901 in MDA-MB-231 cells. Bcl-2 expression decreased in the presence of NS398 up to 24 hours, whereas Bax expression did not change ( Fig. 5B). Therefore, the inhibition of COX-2 in IH-901-treated MDA-MB-231 cells changes the effect of IH-901 on the level of Bcl-2. To more specifically test the requirement for COX-2 induction in the antiapoptotic mechanism, we down-regulated the expression level of COX-2 protein by using the siRNA technique in MDA-MB-231 cells. The target sequences of siRNA used were specific for the COX-2 gene but not the COX-1 gene. We applied adenovirus-mediated delivery of siRNAs strategy to specifically reduce the expression of the COX-2 gene. The adenoviral vectors expressing siRNAs effectively reduced COX-2 gene expression, compared with parental and GFP-siRNA expressing cells, without interrupting ERK 1/2 phosphorylation following treatment with IH-901 ( Fig. 5C). COX-2 knockdown resulted in a dramatic increase in the apoptotic sub-G1 fraction after IH-901 treatment, compared with mock-infected and GFP knockdown ( Fig. 5D). These results show that the COX-2 signaling pathway is critical in blocking IH-901-mediated apoptosis and that COX-2 plays an important role as an antiapoptotic molecule.
Inhibition of COX-2 enzyme activity or knockdown of COX-2 triggered IH-901-mediated apoptosis in MDA-MB-231 and Hs578T cells. A, MDA-MB-231 Cells were pretreated with NSAIDs at the indicated concentration for 30 minutes and then incubated with vehicle or 40 μmol/L IH-901 for 48 hours (top). Hs578T cells were pretreated with NSAIDs for 30 minutes and then incubated with vehicle or 40 μmol/L IH-901 for 24 hours (bottom). The population of cells in sub-G1 phase were measured by propidium iodide staining followed by flow cytometry. Cells with <2 N of DNA content were counted as apoptotic cells and the percentages of apoptotic cells were illustrated graphically. Representative data from three independent experiments. B, MDA-MB-231 cells were pretreated with DMSO vehicle or 250 mmol NS398 for 30 minutes and then incubated with 40 μmol/L IH-901 for the indicated times. Western analyses were done with antibodies specific to Bcl-2, Bax, and α-tubulin. Adenoviral-mediated siRNA for COX-2 efficiently silenced COX-2 expression in MDA-MB-231 cells. After 24 hours of adenovirus-delivered siRNA infection, cells were exposed to either vehicle or 40 μmol/L IH-901 for 10 (C) and 24 hours (D). C, Western blot analyses were done with antibodies specific to phosphorylated forms of ERK and specific to COX-2. Lane 1, mock; lane 2, Ad-siRNA-COX-2 #1; lane 3, Ad-siRNA-COX-2 #2; and lane 4, Ad-siRNA-GFP. D, knockdown of COX-2 by adenoviral-mediated siRNA effectively enhanced the apoptotic fraction of IH-901 treated MDA-MB-231 cells, compared with mock-infected or GFP-siRNA-infected ones. Representative data from three independent experiments. Inset, percentage of sub-G1 phase cells which was determined based on DNA content histogram.
Induction of p27Kip1 by IH-901 Leads to the Inhibition of CDK2 Kinase Activity. Because IH-901 arrested the cell cycle at the G0-G1 phase in MDA-MB-231 and Hep3B cells ( Fig. 2C), we investigated whether IH-901 affects the expressions of G1-specific cell cycle regulatory molecules in MDA-MB-231 cells expressing mutant p53 protein (ref. 34; Fig. 6A ). We examined the protein expression profile of cyclin-dependent kinase inhibitors associated with G1 arrest. Western blot analysis showed that IH-901 treatment of MDA-MB-231 cells strongly induced the protein levels of p27Kip1 in a time-dependent manner, whereas the level of p21WAF1/CIP1 did not change for up to 24 hours. p16INK4a was not detected in MDA-MB-231 or Hs578T cell lines, both of which are homozygously deleted for the INK4 gene (35). The p27Kip1 expression on IH-901 treatment of two cell lines, Hs578T and MKN28, showed similar pattern as in MDA-MB-231 (data not shown). Because the p27Kip1 level was up-regulated in MDA-MB-231 cells by 40 μmol/L IH-901 within 24 hours, we investigated the effect of IH-901 on the kinase activity of CDK2 associated with cyclin E. As shown in Fig. 6B, treatment of MDA-MB-231 cells with IH-901 strongly decreased the histone H1-associated kinase activities of CDK2. The protein levels of cyclin D1 and cyclin E did not change for up to 48 hours after treatment (data not shown). The complexes immunoprecipitated with anti-CDK2 antibody exhibited higher amounts of p27Kip1 from IH-901-treated MDA-MB-231 cells than from mock-treated cells. Next, we investigated the phosphorylation status of the pRb-related proteins pRb/p107 and pRb2/p130. The treatment of MDA-MB-231 cells with 40 μmol/L IH-901 increased the levels of hypophosphorylated forms of pRb and p130 in a time-dependent manner ( Fig. 6C). SNU-16 is a control cell line used as a control for the Rb phosphorylation (27). The increase of p27Kip1 protein levels by IH-901 resulted from the elevation of p27Kip1 mRNA expression ( Fig. 6D, top). We analyzed p27Kip1 mRNA degradation in the absence and presence of IH-901 by Northern analysis, in actinomycin D-treated MDA-MB-231 cells ( Fig. 6D, bottom). The kinetics of p27Kip1 mRNA degradation was similar in both cases, indicating that IH-901 could induce p27Kip1 mRNA at the transcriptional level. The levels of p27Kip1 were also increased in Hep3B following treatment with IH-901 ( Fig. 6A), as observed in MDA-MB-231 cells. These results indicate that in Hep3B cells IH-901 can simultaneously induce p27Kip1-mediated G1 arrest as well as apoptotic cell death without COX-2 induction.
Effect of G1 phase-associated regulatory proteins in MDA-MB-231 cells treated with IH-901. MDA-MB-231 cells were treated with vehicle (ethanol) or 40 μmol/L IH-901 for the indicated times. Cell lysates (30 μg of protein per lane) were resolved by SDS-PAGE, and proteins were detected by immunoblotting with antibodies against the indicated molecules. A, Western blot analysis showed that IH-901 treatment of MDA-MB-231 (top) and Hep3B (bottom) induced the protein levels of p27Kip1. B, IH-901 treatment of MDA-MB-231 cells strongly decreased the histone H1-associated kinase activities of CDK2 in a time-dependent manner (top). The complexes immunoprecipitated with anti-CDK2 antibody exhibited higher amounts of immunodetectable p27Kip1 protein from IH-901-treated cells in a time dependent manner (bottom). C, IH-901 increased hypophosphorylated levels of pRb and p130. SNU-16 is a gastric cancer cell line used as a control for the Rb phosphorylation (27). D, IH-901 treatment elevated the mRNA expression of p27Kip1 (top). p27Kip1 mRNA stability assay (bottom). MDA-MB-231 cells were treated with 40 μmol/L IH-901 for 22 hours, followed by addition of 10 μg/mL actinomycin D at time 0. At the indicated time intervals, total RNAs were isolated and Northern analysis was done. Graph shows the remaining p27Kip1 mRNA quantified after being normalized to corresponding amounts of β-actin mRNA. Points, mean of three separate experiments in duplicate; bars, ± SE.
Ginseng saponins exert various important pharmacologic effects with regard to the control of many diseases including cancer. The present study focuses on identifying the mechanism that underlies the antitumor activity of ginseng saponins. The study shows, for the first time, that IH-901, a novel ginseng saponin metabolite, can cause simultaneous growth arrest and apoptosis. Additionally, adenoviral infection of COX-2 siRNAs provides direct evidence that COX-2 protein can function as an antiapoptotic molecule.
IH-901 could induce the expression of COX-2 protein via ERK signaling. The suppression of IH-901-induced COX-2 expression by the MAP/ERK kinase inhibitor UO126 suggests that the ERK signaling pathway is important in the regulation of COX-2 by IH-901. Knockdown of COX-2 expression by siRNAs did not block ERK activation in MDA-MB-231 cells. IH-901 treatment led to the activation of ERK (data not shown) but failed to induce COX-2 in Hep3B cells. Therefore, IH-901-induced ERK activation is required but not sufficient for inhibition of apoptosis.
Several anticancer drugs have been shown to induce COX-2 expression (22, 23) . For example, microtubule- or actin-interfering agents stimulate MAPK signaling and activator protein activity, enhance PGE2 synthesis, and increase levels of COX-2 mRNA and protein (22). Epigallocatechin-3-gallate was found to up-regulate COX-2 expression and PGE2 production in association with the activation of both the ERK and protein tyrosine phosphatase signaling pathways (36). The suppression of apoptosis by COX-2 is thought to favor carcinogenesis by permitting the survival of cells that have acquired mutations, and is viewed as one of the main mechanisms of tumorigenesis. As COX-2 induction by anticancer drugs may result in unwanted effects such as inflammation, the combined use of selective COX-2 inhibitors and conventional anticancer drugs has been suggested (23).
However, the interpretation of previous NSAID studies was complicated by possible COX-independent mechanisms. In many cases, NSAIDs alone induced apoptosis at much higher concentrations than their IC50's. COX-2 mutant proteins lacking COX activity have been reported to inhibit cell cycle progression in a variety of cell types (37). In order to confirm that COX-2 protein is an antiapoptotic molecule, we constructed adenoviral COX-2 siRNAs for selective targeting of the COX-2 protein itself rather than opting to use NSAIDs to inhibit COX-2 enzymatic activity. Our data shows that both NSAID treatment and knock-down of the COX-2 gene by siRNAs could cause apoptosis in MDA-MB-231 cells, providing direct evidence that COX-2 protein plays an important role in suppressing IH-901-mediated apoptosis.
Thus far, the antitumor activity of IH-901 has been attributed solely to apoptosis (8, 9) . However, in this study, we show a second mechanism: cell cycle arrest. IH-901 strongly induced p27Kip1 and inhibited CDK2 activities followed by an increase in hypophosphorylated levels of pRb and p130. These molecular effects of IH-901, especially the up-regulation of p27Kip1, may play important roles in the G1 arrest seen in MDA-MB-231, MKN28, Hs578T, and Hep3B cells. The observed up-regulation of p27Kip1 by IH-901 and resulting decrease of CDK2 kinase activity is important and consistent with an earlier report that decreased p27Kip1 expression in breast carcinomas are associated with aggressive phenotypes and that adenoviral infection with human p27Kip1 resulted in cell cycle arrest (38). The pRb-related proteins, p107 and p130, cooperate to regulate cell cycle progression at G1-S checkpoint. It has been reported that p130 and p27Kip1 are mutually involved in the negative regulation of cellular proliferation (39). IH-901 reduced CDK2-cyclin E kinase activity, and increased the hypophosphorylated levels of Rb-related proteins. Hep3B is known to be Rb-negative (40) and has mutant p53 (41). Even though Hep3B cells showed marginal G1 arrest, the progressive decline of the S phase was indicative of G1 arrest ( Fig. 2C). However, it is difficult to exclude the possibility of the involvement of p21WAF1/CIP1 in IH-901-mediated growth arrest because all four cell lines used in this study express mutant p53. We found the induction of p21WAF1/CIP1 mRNA and protein in a carcinoma cell line containing wild-type p53 after the treatment with IH-901 (data not shown). Collectively, the growth inhibitory effects of IH-901 in cells could be caused by the following progression: induction of p27Kip1 leads to a decrease in kinase activity of CDKs followed by modulation of further downstream targets such as pRb-related proteins and the E2F family of transcription factors family.
In summary, the findings of this study show that IH-901, a novel ginseng saponin metabolite, induces apoptosis and inhibits cell proliferation by G1 arrest via increase of p27Kip1 expression. COX-2 induction caused by IH-901 could diminish the antitumor effects, and strongly suggests the therapeutic use of COX-2 siRNAs to inhibit COX-2. Further study will contribute to the development of IH-901 or ginseng-related drugs as potential chemotherapeutic or chemopreventive agents.
Grant support: Supported in part by the National Cancer Control R&D Program 2003, Ministry of Health and Welfare, Republic of Korea; by Seoul National University Cancer Research Institute; and by the 2002 BK21 Project for Medicine, Dentistry, and Pharmacy.
We thank the Il Hwa Company for providing IH-901, Drs. S.M. Prescott for cDNA construct of COX-2 and J-W. Soh for his helpful discussion, and to J.S. Koh for his critical editing of this manuscript.
Revision received November 16, 2004.
Accepted December 29, 2004.
Yun TK. Experimental and epidemiological evidence on non-organ specific cancer preventive effect of Korean ginseng and identification of active compounds. Mutat Res 2003; 523-524: 63–74.
Yun TK, Choi SY, Yun HY. Epidemiological study on cancer prevention by ginseng: are all kinds of cancers preventable by ginseng? J Korean Med Sci 2001; 16 Suppl: S19–27.
Shin HR, Kim JY, Yun TK, Morgan G, Vainio H. The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence. Cancer Causes Control 2000; 11: 565–76.
Hasegawa H, Sung JH, Matsumiya S, Uchiyama M. Main ginseng saponin metabolites formed by intestinal bacteria. Planta Med 1996; 62: 453–7.
Hasegawa H, Sung JH, Matsumiya S, et al. Reversal of daunomycin and vinblastine resistance in multidrug-resistant P388 leukemia in vitro through enhanced cytotoxicity by triterpenoids. Planta Med 1995; 61: 409–13.
Lee SJ, Sung JH, Lee SJ, Moon CK, Lee BH. Antitumor activity of a novel ginseng saponin metabolite in human pulmonary adenocarcinoma cells resistant to cisplatin. Cancer Lett 1999; 144: 339–43.
Lee BH, Lee SJ., Hur JH, et al. In vitro antigenotoxic activity of novel ginseng saponin metabolites formed by intestinal bacteria. Planta Med 1998; 64: 500–3.
Lee SJ, Ko WG, Kim JH, Sung JH, Moon CK, Lee BH. Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of caspase-3 protease. Biochem Pharmacol 2000; 60: 677–85.
Choi HH, Jong HS, Park JH, et al. A novel ginseng saponin metabolite induces apoptosis and down-regulates fibroblast growth factor receptor 3 in myeloma cells. Int J Oncol 2003; 23: 1087–93.
Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci 2003; 24: 96–102.
Wang D, Dubois RN. Cyclooxygenase-2: a potential target in breast cancer. Semin Oncol 2004; 31: 64–73.
Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994; 107: 1183–8.
Lim HY, Joo HJ, Choi JH, et al. Increased expression of cyclooxygenase-2 protein in human gastric carcinoma. Clin Cancer Res 2000; 6: 519–25.
Shim V, Gauthier ML, Sudilovsky D, et al. Cyclooxygenase-2 expression is related to nuclear grade in ductal carcinoma in situ and is increased in its normal adjacent epithelium. Cancer Res 2003; 63: 2347–50.
Yoshimatsu K, Altorki NK, Golijanin D, et al. Inducible prostaglandin E synthase is overexpressed in non-small cell lung cancer. Clin Cancer Res 2001; 7: 2669–74.
Fujita H, Koshida K, Keller ET, et al. Cyclooxygenase-2 promotes prostate cancer progression. Prostate 2002; 53: 232–40.
Chang SH, Liu CH, Conway R, et al. Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc Natl Acad Sci U S A 2004; 101: 591–6.
Pai R, Nakamura T, Moon WS, Tarnawski AS. Prostaglandins promote colon cancer cell invasion; signaling by cross-talk between two distinct growth factor receptors. FASEB J 2003; 17: 1640–7.
Cao Y, Prescott SM. Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol 2002; 190: 279–86.
Nzeako UC, Guicciardi ME, Yoon JH, Bronk SF, Gores GJ. COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cells. Hepatology 2002; 35: 552–9.
Sun Y, Tang XM, Half E, Kuo MT, Sinicrope FA. Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells. Cancer Res 2002; 62: 6323–8.
Subbaramaiah K, Hart JC, Norton L, Dannenberg AJ. Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 AND p38 mitogen-activated protein kinase pathways. J Biol Chem 2000; 275: 14838–45.
Hsueh CT, Chiu CF, Kelsen DP, Schwartz GK. Selective inhibition of cyclooxygenase-2 enhances anticancer drug-induced apoptosis. Cancer Chemother Pharmacol 2000; 45: 389–96.
Milas L, Hanson WR. Eicosanoids and radiation. Eur J Cancer 1995; 31A: 1580–5.
Polyak K, Kato JY, Solomon MJ, et al. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-β and contact inhibition to cell cycle arrest. Genes Dev 1994; 8: 9–22.
Rao S, Gray-Bablin J, Herliczek TW, Keyomarsi K. The biphasic induction of p21 and p27 in breast cancer cells by modulators of cAMP is posttranscriptionally regulated and independent of the PKA pathway. Exp Cell Res 1999; 252: 211–23.
Kim SG, Kim SN, Jong HS, et al. Caspase-mediated Cdk2 activation is a critical step to execute transforming growth factor-β1-induced apoptosis in human gastric cancer cells. Oncogene 2001; 20: 1254–65.
Jong HS, Lee HS, Kim TY, et al. Attenuation of transforming growth factor β-induced growth inhibition in human hepatocellular carcinoma cell lines by cyclin D1 overexpression. Biochem Biophys Res Commun 2002; 292: 383–9.
Kim SG, Jong HS, Kim TY, et al. Transforming growth factor-β1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells. Mol Biol Cell 2004; 15: 420–34.
Song SH, Jong HS, Choi HH, et al. Transcriptional silencing of cyclooxygenase-2 by hypermethylation of the 5′ CpG island in human gastric carcinoma cells. Cancer Res 2001; 61: 4628–35.
Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–3.
Saha D, Datta PK, Sheng H, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor-β1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1999; 1: 508–17.
Fan XM, Wong BC, Lin MC, et al. Interleukin-1β induces cyclo-oxygenase-2 expression in gastric cancer cells by the p38 and p44/42 mitogen-activated protein kinase signaling pathways. J Gastroenterol Hepatol 2001; 16: 1098–104.
Negrini M, Sabbioni S, Haldar S, et al. Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions. Cancer Res 1994; 54: 1818–24.
Musgrove EA, Lilischkis R, Cornish AL, et al. Expression of the cyclin-dependent kinase inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer. Int J Cancer 1995; 63: 584–91.
Park JW, Choi YJ, Suh SI, Kwon TK. Involvement of ERK and protein tyrosine phosphatase signaling pathways in EGCG-induced cyclooxygenase-2 expression in Raw 264.7 cells. Biochem Biophys Res Commun 2001; 286: 721–5.
Trifan OC, Smith RM, Thompson BD, Hla T. Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism. J Biol Chem 1999; 274: 34141–7.
Craig C, Wersto R, Kim M, et al. A recombinant adenovirus expressing p27KIP1 induces cell cycle arrest and loss of cyclin-Cdk activity in human breast cancer cells. Oncogene 1997; 14: 2283–9.
Howard CM, Claudio PP, De Luca A, et al. Inducible pRb2/p130 expression and growth-suppressive mechanisms: evidence of a pRb2/p130, p27KIP1, and cyclin E negative feedback regulatory loop. Cancer Res 2000; 60: 2737–44.
Hashimoto O, Ueno T, Kimura R, et al. Inhibition of proteasome-dependent degradation of Wee1 in G2-arrested Hep3B cells by TGF-β1. Mol Carcinog 2003; 36: 171–82.
Lin Y, Shi CY, Li B, et al. Tumour suppressor p53 and Rb genes in human hepatocellular carcinoma. Ann Acad Med Singapore 1996; 25: 22–30.
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Cancer Res March 1 2005 (65) (5) 1952-1960; DOI: 10.1158/0008-5472.CAN-04-1740
Inactivation of the Mitogen-Activated Protein Kinase Pathway as a Potential Target-Based Therapy in Ovarian Serous Tumors with KRAS or BRAF Mutations
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Tag Archives: Partners in Crime
Agatha Christie Ltd CEO Hilary Strong talks to DQ
December 1, 2015 One on oneAgatha Christie Ltd, And Then There Were None, BBC1, Endor Productions, Hilary Strong, Kenneth Branagh, Lifetime, Mammoth Screen, Murder on the Orient Express, Partners in Crime, Sarah PhelpsMichael Pickard
Agatha Christie Ltd CEO Hilary Strong explains why adaptations of the celebrated author's stories, which remain popular across the world, will keep on coming.
The 125th anniversary of the birth of author Agatha Christie this year is being marked with two new television adaptations.
Sleuthing couple Tommy and Tuppence appeared in a new BBC1 series called Partners in Crime, starring David Walliams and Jessica Raine, in July. A six-part drama placing the characters in the 1950s, it re-imagined the events of Christie novels The Secret Adversary and N or M? across two three-part stories produced by Endor Productions.
BBC1 has also partnered with US cable network Lifetime on a new adaptation of And Then There Were None, which was named the world's favourite Christie novel in a survey published in September.
And Then There Were None will air on BBC1 this Christmas
The classic thriller, which tells of 10 individuals invited to an isolated island where they are killed one by one by an unknown murderer, has been adapted by Sarah Phelps (Great Expectations) and produced by Mammoth Screen. The cast includes Douglas Booth, Charles Dance, Anna Maxwell Martin, Sam Neill, Miranda Richardson, Toby Stephens and Aidan Turner, and is due to air on BBC1 this Christmas.
These new adaptations serve as a fitting tribute to the prolific writer, dubbed the Queen of Crime. But they also represent the efforts of Agatha Christie Ltd to introduce her to a new generation of fans by becoming more proactive when exploiting the rights to the author's vast library.
Hilary Strong (pictured top), CEO of Agatha Christie Ltd, says: "The brief from Mathew Pritchard, Christie's only grandchild and chairman of the company, was that we would work together to exploit the brand ourselves and that's why he brought me in with my television background," explains Strong. "We were coming to the end of our Poirot films series on ITV that first aired in 1989. David Suchet's work as Poirot is iconic and no one else has played a leading drama role for 25 years. It's an extraordinary thing to have done but we knew they were finishing and we knew then that we had to do something new and fresh.
"It gave us the opportunity to sit back and decide what we wanted to do. Did we want to carry on with Christie being the traditional, much-loved work that it is? People's perception of it is 'cosy crime.' But for a brand to remain alive and resonate for a modern audience, it needs to do something new and give a different message."
With a background in television and rights management, Strong is perfectly placed for this new challenge. She was previously MD of Acorn Productions, where she had responsibility for developing drama around the works of Christie and other properties, including Foyle's War. She has also worked for Chorion (Enid Blyton, Paddington Bear) and was group business director at Hat Trick Productions.
Partners in Crime, starring David Walliams and Jessica Raine
With Partners in Crime, the company made its first move away from 'cosy crime,' setting the action in a more recent period and casting Walliams and Raine to attract a younger audience. And Then There Were None, while set in 1939, has a more contemporary tone and is, Strong says, "really fucking scary."
She explains: "Sarah Phelps has done an amazing job and has been absolutely truthful to the story while giving us this deep dark tone. For me, it embodies what we've been trying to do – take something and retell it so it appeals to modern audiences. If we can achieve that, then we've done our job. I don't want to shake off the cosy crime image but I want people to understand that Christie can be delivered in a different way."
The company isn't just interested in a new way of telling Christie – it is also shaking up the way its television adaptations are built. No longer simply licensing rights away, Agatha Christie Ltd is keeping its hand in the creative process and building direct relationships with broadcasters and suggesting potential projects before selecting the production partners they want to work with to bring the idea to the screen.
"It is quite unusual," Strong says of the strategy. "It helps that there are relationships before that process. Damien Timmer, who runs Mammoth, was an executive producer at ITV on Poirot and Miss Marple, so he has a long relationship with the family and the company. When we sat down with him, he was extremely open to the benefits of collaboration because you get a different insight when you've got people who really know the brand involved. The script process is very collaborative but once production starts, they get on and make the programme. It works really well.
"The thing I was most keen to do was move away from the idea that the estate is there to approve or disapprove, which does happen with estates. So if we get it right and we've chosen the right writer and worked on the scripts, then once you get to production, those parts are in place. It would be quite unusual to hit a fundamental problem then.
"We also do a lot of the design stuff together because that's really important to us. We need to make sure the imagery being used when you get to promotion works cross-platform. We had new book covers for Partners in Crime, with a logo that goes across the TV programmes."
France 2's Les Petits Meutres d'Agatha Christie
Christie isn't just popular in Britain, however. Strong says the novelist's works have been translated into more languages than those of any other author, while TV adaptations have been sold into more than 180 countries. Japanese network NHK aired a two-part version of Murder on the Orient Express in January this year, produced by Fuji TV. And French broadcaster France 2 is behind Les Petits Meutres d'Agatha Christie, which plants two detectives into Christie plots. Twenty-three episodes have been produced by Escazal Films since 2009.
"What's been really interesting is just how big Christie is in other countries," says Strong. "In South America, Australia, Germany, Italy, Japan and China, Christie is huge and they have their own indigenous productions in foreign languages."
She also suggests a big deal for the German-language rights to Christie's books is near completion, adding that the estate is very open to doing "very radical, avant garde, contemporary new things" with Christie's stories.
"But we're unlikely to muck about with the core plot because that's what works," she explains. "When people start trying to mess with it, that's when it goes wrong. You can tell it in a new way, give it a contemporary tone or set it in a contemporary setting. And Then There Was None means there is no one left at the end. It's not a returning series!"
Strong recognises the drama business is tougher now than at any point in her career: "The fact that budgets have come down and expectations in terms of quality are higher, together with the need to compete on the international market, means your vision and scale has had to go up.
"It's a very good time for drama. There's an awful lot out there but that's because there's an appetite for it. As people keep on watching it, people want more. And the fact there's so much, if you've got a brand like Christie, you can put your head above the parapet a bit and people can find you in the schedules.
The Japanese version of Murder on the Orient Express
"But one of the things we don't do is work with people just because they think the brand will help them sell more shows. We will only work with people we know have a genuine love for the stories. We have tried developing a couple of smart ideas and then down the road realised the people we were working with didn't have the depth of understanding of the brand, and in those circumstances it rarely works. If you work with people who understand Christie, it just works much better."
Strong would love to see Witness for the Prosecution, a short story about a woman who gives evidence for the prosecution in her husband's murder trial, made for television and says the global appeal of Christie's stories is in the pure and simple language she uses.
In the meantime, fans can look forward to a new big-screen version of Murder on the Orient Express, which will be directed by Kenneth Branagh for 20th Century Fox. Branagh will also star as Belgian detective Hercule Poirot, who must investigate a murder on board the famous train – but there are a number of passengers who could have committed the crime.
Agatha Christie Ltd has also launched its own digital drama based upon Christie's character Mr Quin, which launched as an app in November 2015.
But what is it about Christie's work that means it has stood the test of time? "Her plot lines are just ingenious and her characters are lovable," Strong adds. "People adore them. And the breadth – there are 33 Poirot novels to read. You're not going to do it in a hurry.
"I don't see any time when people don't want to carry on reading her books. Our job is to retell those stories in a way that makes them accessible for people. What I'd love – and would tell me I'd done my job – is if people watched And Then There Were None and then went back and read some of the original books."
tagged in: Agatha Christie Ltd, And Then There Were None, BBC1, Endor Productions, Hilary Strong, Kenneth Branagh, Lifetime, Mammoth Screen, Murder on the Orient Express, Partners in Crime, Sarah Phelps
Disney's Descendants on the up
August 7, 2015 Hit & MissAmazon, And Then There Were None, BBC1, BBC2, Cedar Cove, Channel 5, Descendants, Disney, Hallmark Channel, Kenny Ortega, Michelle Vicary, NBC, Odyssey, Partners in Crime, Sneaky Pete, Spike, TutAndy Fry
Descendants was directed by High School Musical's Kenny Ortega
When Disney gets it right, it really gets it right. Currently doing great business for Disney Channel US is Descendants, a modern-day story based around the teenage offspring of Disney's most notorious villains. Having attracted 6.6 million viewers for its premiere, the live-plus-three-day audience for the show increased to 10.5 million.
Those figures make Descendants the number-one cable TV movie of 2015, which is perhaps not so surprising when you learn that it was directed, choreographed and exec produced by High School Musical's Kenny Ortega. HSM was a huge franchise, spawning three movies and giving the world the adorable Zac Efron.
Descendants is also performing well across other platforms. It currently holds the top spot on iTunes (Top TV seasons), while its soundtrack is at the top of the iTunes soundtrack chart and third on the iTunes album chart. It has also spawned a best-selling prequel novel, The Isle of the Lost by Melissa de la Cruz, and an animated shortform series, Descendants Wicked World.
More movie-length productions are presumably an option, although the concept and characterisation might also lend itself to a live-action series.
Cedar Cove stars Andie MacDowell (left)
Hallmark Channel is also celebrating this week following a strong showing from Debbie Macomber's Cedar Cove, a romantic drama starring Andie MacDowell and Dylan Neal.
According to Hallmark, the latest episode of Cedar Cove became the network's most watched and highest rated among total viewers and households in its Saturday 20.00 time period. To date, season three of the show is averaging a 2.0 household rating and two million total viewers.
"Over the last four years, Hallmark Channel's scripted series have become appointment viewing for our audience," said Michelle Vicary, executive VP for programming and network publicity at Hallmark owner Crown Media Family Networks. "The popularity of Cedar Cove, Good Witch and When Calls the Heart demonstrate the power of our brand and the resonance of our storytelling."
With so much competition in scripted content, a lot of launch success these days is down to whether you can offer something that piques the audience's interest.
Breaking Bad's Bryan Cranston in Sneaky Pete
Today, for example, Amazon launches Sneaky Pete, the pilot for a conman drama written and executive produced by David Shore and Bryan 'Walter White' Cranston.
Backed by Sony, the show was originally intended for CBS. But when the network withdrew its interest, Amazon stepped in and greenlit the pilot. The star of the pilot is Giovanni Ribisi, but Cranston will guest star, which is sure to lure in swathes of Breaking Bad fans.
That might be enough to convince Amazon it is worth going to series with Sneaky Pete – particularly if it results in new subscribers for the service.
Amazon's approach is to show pilots for free and then base its commissioning decisions on audience data and customer feedback. Alongside Sneaky Pete, another pilot hopeful launching today is Casanova, from Jean Pierre Jeunet, Stuart Zicherman and Electus's Ben Silverman.
The Guardian said Odyssey 'failed to produce a single moment of originality'
In previous weeks, we've reported that ancient Egypt drama Tut achieved good ratings for its launch episode on cable channel Spike in the US. It has now aired as a two-part miniseries on Channel 5 in the UK.
C5, which like Spike is owned by Viacom, attracted around 880,000 viewers across two episodes. This is around 28% above the slot average. With Spike having announced its intention to invest more money in scripted shows, Channel 5 may find itself a long-term beneficiary of this.
A less successful acquisition has been thriller series Odyssey, snapped up by BBC2 at the LA Screenings this year after an initial airing on NBC in the US. In this show, British actress Anna Friel plays US army special ops member Odelle Ballard, who is the sole survivor of a drone attack in Mali but has been reported dead after her team discovers a US terrorism funding conspiracy.
On paper, Odyssey looked like it might be a combination of Homeland and The Honourable Woman, but it has received poor reviews and ratings. Already axed by NBC in the US, it has seen its audience on BBC2 slide from around 2.5 million at the start to around one million at the end of its run.
Friel's acting was praised, but The Guardian summed up the general mood among critics: "Why beat around the bush with subtlety when each coarsely drawn character can spell out plot and motivation so clearly and deliberately? You can practically hear the clunk and ding of the typewriter whenever someone opens their mouth. Even the secret documents kept safe in a USB stick around (Ballard's) neck are written in Fisher-Price spy language. The writers do not believe you'll keep up any other way."
While BBC2 has had a summer to forget regarding drama, BBC1 continues to do pretty well with its Agatha Christie adaptation Partners in Crime. The second episode of six attracted five million viewers last Sunday evening.
While this is down from episode one (6.5 million), it's still pretty respectable. It bodes well for another upcoming Christie adaptation based on iconic novel And Then There Were None. Due to air later this year, And Then There Were None is a Mammoth Screen and Agatha Christie Productions programme for the BBC, coproduced with A+E Television Networks. RLJ Entertainment has taken US DVD and DTO rights, while A+E Networks will handle international sales via A+E Studios International.
tagged in: Amazon, And Then There Were None, BBC1, BBC2, Cedar Cove, Channel 5, Descendants, Disney, Hallmark Channel, Kenny Ortega, Michelle Vicary, NBC, Odyssey, Partners in Crime, Sneaky Pete, Spike, Tut
Will C4's robots return?
July 31, 2015 Hit & MissAMC, Bajo el Mismo Cielo, Banijay, BBC, Channel 4, Humans, Lilyhammer, Mina Lefevre, MTV, Partners in Crime, Penny Dreadful, Relativity Media, Ryan Kavanaugh, Scream, Showtime, Sky Atlantic, Telemundo, Tierra de Reyes, Univision, Welcome to Sweden, ZodiakAndy Fry
Emily Berrington as a 'Synth' in Humans
The finale of Humans' first season airs in the UK this weekend and the show continues to do exceptionally well for Channel 4. BARB ratings for the first six episodes show there was an inevitable dip after stellar ratings for the first two parts, but that the show has stayed remarkably consistent since then. From episodes three to six, it recorded between 3.63 million and 3.93 million viewers (seven-day figures) – way ahead of anything else on C4.
With the show now approaching its climax, it would be a major surprise if it didn't equal or surpass those figures for episodes seven and eight. And then a commission for a second season would look highly likely.
The only cloud on the horizon for Humans is that AMC in the US is not getting such good ratings with the series (which may place a question mark over its involvement in a second season), but the strength of the C4 showing ought to be enough to see it through.
Still in the UK, there was a strong showing for BBC1's Agatha Christie adaptation Partners In Crime, which attracted 6.5 million viewers for its first episode (Sunday at 21.00). Starring David Walliams and Jessica Raine as amateur detectives Tommy and Tuppence, the six-part show is the channel's biggest new drama launch since Poldark, demonstrating that Sunday evening is still a time when audiences like to spend time with familiar faces and brands.
Partners in Crime opened strongly for BBC1 on Sunday evening
Earlier in July, Sky Atlantic and Showtime announced plans for a third season of gothic horror drama Penny Dreadful. Looking at the final ratings for season two of the show on Sky Atlantic, it's easy to see why. According to BARB's seven-day data, the final episode attracted an audience of 544,000 – up from 450,000-500,000 for the previous few episodes.
With Game of Thrones finished for another year, Penny Dreadful became channel's top-performing drama, some way ahead of The Affair (433,000) and True Detective season two (352,000). In the week following Penny Dreadful's departure, nothing on Sky Atlantic managed to attract more than 315,000 viewers. The show has also been attracting attention internationally, securing a deal with Australian subscription VoD platform Presto last week.
In the US, MTV is halfway through the first 10-part season of Scream, a horror series that has been spun out of the iconic feature-film franchise. A strong debut saw the show attract six million viewers (live plus three), making it "the most watched new series premiere of the summer on cable with millennials," according to MTV. In an added bonus, the first episode was also streamed more than 500,000 times on MTV.com and the MTV app.
Scream will get a second season after performing well on MTV
Since then the ratings have dropped a little but stayed strong enough for MTV to announce a second season. At the midway point, 21 million viewers have tuned in to Scream on air while the series has generated 7.9 million streams across other platforms.
Speaking at the Television Critics Association's summer event this week, MTV head of scripted programming Mina Lefevre said: "It has been a wonderful experience working with (Scream exec producer) Bob Weinstein and his team, who are such connoisseurs of this genre, and we are thrilled by how viewers have responded to the reinvention of Scream."
Meanwhile, the US TV industry's love affair with Scandinavia took a double hit this week. Following the news that Netflix has cancelled Lilyhammer, NBC announced that it has canned eOne's low-rated comedy Welcome to Sweden. The show, created by Greg Poehler, did moderately well in season one but has fallen away badly in season two, with NBC pulling it from the air after just four editions of its 10-episode run.
Welcome to Sweden has been cancelled soon into its second run
Commenting on Instagram, Poehler said: "Due to some craptastically low ratings in the US, WTS is officially done. I am eternally grateful to all of our fans. When you make a show – and write, produce, obsess and act in it – all you want is for someone, somewhere, to tell you they appreciate it. There have been so many of you in both Sweden and the US that have done so, and every compliment has made me immeasurably happy. So, thank you. Thank you, thank you…"
In the Hispanic US market, Telemundo continues to make inroads into the audience share of its major competitor Univision. DQ reported on the success of El Senor de los Cielos last week, and now Telemundo says the finale of Tierra de Reyes (Land of Honor) attracted 2.39 million total viewers.
This helped make Telemundo the number-one Spanish-language network in primetime, beating Univision. It has also just launched Bajo el Mismo Cielo (Under the Same Sky), the story of a hard-working Mexican immigrant who crosses the border illegally and settles with his family in LA. Episode one attracted 1.72 million viewers and also hit 3.3 million global Facebook users with a 30-minute preview.
On the corporate front, the TV market is waiting to see the implications of the US$1bn merger between Banijay Group and Zodiak Media (announced this week). Between them, the two companies own approximately 45 prodcos.
Tierra de Reyes has helped Telemundo make up ground against Univision
While there are some complementary areas between the two businesses, there is also a lot of overlap in markets like the US, France and Scandinavia. In drama terms, the deal brings together companies including Touchpaper (UK), Yellow Bird (Scandinavia), Magnolia (Italy), Marathon Media (France), DLO Producciones (Spain) and Screentime (Australia and New Zealand).
Finally, this week's big corporate 'miss' is Ryan Kavanaugh's film and TV studio Relativity Media, which has filed for bankruptcy after racking up $320m in unpaid loans. Kavanaugh's next-generation studio model, with its strong emphasis on data analysis, enthralled the industry for a few years but in the end couldn't survive a number of high-profile box-office failures.
Speaking at Mipcom in 2013, Kavanaugh expressed his intention to get more into the TV business, expounding his theory that films "are perhaps the greatest TV pilots ever." His goal at that stage was to sell five or six TV series properties a year. However, this never came to pass. The main TV titles to come out of Relativity have been National Geographic's Act of Valor and Limitless, a spin-off of the Bradley Cooper-starring movie in the form of a series will soon air on CBS.
tagged in: AMC, Bajo el Mismo Cielo, Banijay, BBC, Channel 4, Humans, Lilyhammer, Mina Lefevre, MTV, Partners in Crime, Penny Dreadful, Relativity Media, Ryan Kavanaugh, Scream, Showtime, Sky Atlantic, Telemundo, Tierra de Reyes, Univision, Welcome to Sweden, Zodiak
BBC partners Agatha Christie to bring crime caper to TV
July 3, 2015 IN FOCUSAgatha Christie, BBC1, David Walliams, Edward Hall, Endor Productions, Jessica Raine, Partners in CrimeMichael Pickard
DQ takes a closer look at the forthcoming BBC1 show Partners in Crime, starring David Walliams and Jessica Raine and based on a series of novels by celebrated author Agatha Christie.
In the crowded world of TV detectives, a new drama featuring the crime-solving capers of a married couple has ambitions to bring a mix of thrills and humour to the genre.
Set in a 1950s Britain thrust from the aftermath of the Second World War into the early throes of a new Cold War, BBC1's forthcoming series Partners in Crime (main image) sees husband-and-wife team Tommy and Tuppence inject some adventure into their marriage when they stumble their way into a world of murder and conspiracies.
David Walliams (right) came up with the idea of bringing the story to TV
Described as Agatha Christie meets Indiana Jones, the six-part series is based on the celebrated author's first two full-length novels to feature the pair – The Secret Adversary and N or M?
Tommy and Tuppence are played by David Walliams and Jessica Raine respectively, and it was Walliams who first approached Agatha Christie Productions with the idea of bringing the couple to the small screen. Once the BBC was on board, Endor Productions was signed up to deliver the series, which is distributed by RLJ Entertainment.
Little Britain star Walliams says: "I've been a fan of Agatha Christie since I saw the movie of Murder on the Orient Express as a kid, when I was about eight, and I was completely blown away by the story and haunted by it for a long time afterwards.
"I've read a lot of Christie's work and recognised these characters hadn't been done for quite a while – there was a TV series in the 1980s but in recent times there hadn't been many adaptations, and I thought there was a good opportunity to have a new version.
"There was something for me that really appealed about a husband-and-wife detective duo, and I thought there was something really delicious at the centre of it. You'd have Agatha Christie's brilliant box of adventures but at the centre of it you'd have a human story. These are characters who are not genii in the way of a lot of book detectives like Sherlock Holmes or Poirot – these were normal people in that situation."
The Secret Adversary was Christie's second novel, published in 1922 after The Mysterious Affair at Styles, which first introduced readers to Belgian detective Hercule Poirot. Tommy and Tuppence then appeared in a further four stories through Christie's career.
"My grandmother was very fond of them," says Mathew Prichard, Christie's grandson and chairman of Agatha Christie Ltd. "They featured in her second book and, 50 years later, in her last book. Occasionally throughout her career, when she felt she needed a rest and she needed to lose herself in something, she wrote about Tommy and Tuppence. The Secret Adversary, the first story we're treating, came in 1922, and N or M? in the middle of the Second World War in the 1940s.
"Tommy and Tuppence represent something entirely different from Poirot or Miss Marple. They represent a partnership, and the most important thing for me is that when I watch them, they are intrinsically Agatha Christie. They are very much the person that I knew in the 1950s, where these films are set, and I can't help watching them with a considerable sense of nostalgia, affection and recognition of the person whom I respected and loved so much. Dare I say it, I hope we do some more."
The TV adaptation, timed to coincide with the 125th anniversary of Christie's birth, plants the action in the 1950s, bringing together elements of the Tommy and Tuppence novels that span half a century.
And for both Walliams and Raine, best known for her work in Call the Midwife and Fortitude, it was the relationship between the crime-solving couple that attracted them to the project. While Tuppence is a woman who sees adventure round every corner, her husband is decidedly more cautious.
Walliams says: "I like that Tommy has to defer to Tuppence, which I think a lot of viewers will recognise, and it will have a lot of appeal that the woman is in charge. The woman is definitely running the show and she's more intelligent and heroic than he is.
"The idea of Tuppence saving the day and being the more forthright, heroic one is definitely there (in the books), and she's the one yearning for adventure. We definitely wanted to be true to the spirit of it. I'm the damsel in distress."
Raine says the "team element" was key to her taking on the role of Tuppence. "It was the way they were equal. There's not really any sidekick element. I really liked how front-footed she is, how quick-witted. She's quite funny, intelligent. She's all of these things you want to be in real life, and I liked how Tommy is more on the back foot, while Tuppence is quite nosey and curious. Putting characters in alien situations is always very attractive because you get to play that. It was irresistible as soon as I read the script. There's no hint of sadness, she's a very confident woman. It was really refreshing – she's incredible."
Jessica Raine (left) in Call the Midwife
With a host of TV crime dramas focusing on the hunt for serial killers who prey on women, both Raines and Partners in Crime director Edward Hall said it was pleasing to portray a woman who wasn't playing the victim.
"Tuppence is really modern for a woman in the 50s," says Raine. "She was, I felt, a little frustrated at where her life had got to. I don't think of her as a typical woman of the 50s, if there is such a thing. It was so nice to play a woman who isn't in any way put-upon or a victim. That was a massive appeal for me."
Hall adds: "It's very hard to find heroines in TV drama who are heroines for reasons other than overcoming some kind of physical or sexual violence, or something else that makes them a victim. You very rarely see women coming in and saving the day. I thought that was a particularly good thing about this project."
Unlike so many crime dramas on television, Partners in Crime seems to slip effortlessly between tension-filled scenes and the touches of comedy rooted in Tommy and Tuppence's relationship, which are heightened by Walliams's comedy instincts.
"What we were trying to achieve in the making of this is a swing in tone from thriller to high comedy sometimes, wrapped up with all the characteristic joys of a good Agatha Christie story," Hall explains. "It all starts and finishes with the scripts. We had some fantastic scripts from Claire (Wilson, who adapted N or M?) and Zinnie (Harris, who wrote The Secret Adversary), and that guides you.
"If something had a degree of jeopardy, you wanted to make it as scary as possible so that it felt real. And if something was funny, you could swing back at the drop of a hat. There's a moment in episode five when Jessica has a farce moment with a maid in a corridor, trying to get to a room. The music's quite fun, it's quite a funny moment. She gets to the door, she opens the door, and then the atmosphere completely changes when she walks into this room. She's not meant to be there, it's very serious – it's life and death. We just tried to be alive to those changes in tone."
Executive producer Hilary Bevan Jones, founder of Endor Productions, said the characters' high energy and spirit was key to the way the series was conceived as "Agatha Christie meets Indiana Jones."
Hilary Strong, CEO of Agatha Christie Ltd, adds: "A great story is a great story, whenever it's told, and I think people forget, because Christie's stories have been around a long time, that she was a really contemporary writer. She was a very contemporary woman of her time.
"One of the fantastic things about Agatha Christie is that her work has always really appealed to young readers. With this piece of work we wanted to get back to that, to have something that's useful and fun and where people could really see the joy in Christie's work with those great storylines underneath. Tommy and Tuppence is the perfect way to refresh, to bring Christie back."
But what would the celebrated author herself think of this new take on Tommy and Tuppence, which will launch on BBC One later this month?
"I'm sure she would have loved it," says Prichard. "The uniqueness of this project is that you're almost seeing my grandmother at a certain age on the screen. There's all the humour, energy and vitality, and it really isn't like Poirot, even though she wrote a Poirot book a year before.
"This is much more natural, much more realistic. We all know a Tommy and Tuppence, but we don't know a Poirot."
tagged in: Agatha Christie, BBC1, David Walliams, Edward Hall, Endor Productions, Jessica Raine, Partners in Crime
Besa foot forward
Doing more with Les
Mysterious Girl
Hair to die for
On the ward

redpajama_set_name: RedPajamaCommonCrawl


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NFL.com: Marlon Mack Among NFL's Most Explosive Runners
After reaching his first 1,000-yard season in 2019, NFL.com has named Indianapolis Colts running back Marlon Mack as one of the league's most-explosive runners.
Jake Arthur
Colts.com Contributor
INDIANAPOLIS — In 2019, the Indianapolis Colts had one of their best seasons running the football since before many of their fans were even born.
They had the seventh-most rushing yards in the NFL at 133.1 yards per game, they had the 10th-most run plays of at least 10 yards with 53, they had their fifth-best yards-per-carry average in franchise history at 4.52, and they eclipsed 2,000 rushing yards in a season for the first time since 1994.
Their rushing attack's top dog was Marlon Mack, who became one of the league's most productive runners en route to a career year.
Recently, Nick Shook of NFL.com evaluated which players in the NFL are the most explosive runners based on a minimum of 100 carries in 2019, at least 20 carries of 10-plus yards, and a certain percentage of their runs reaching at least 15 miles per hour.
Mack claimed a spot on the list, landing at No. 7, above players like Todd Gurley, Christian McCaffrey and Chris Carson. Shook writes:
7. Marlon Mack
Runs of 10-plus yards: 28
10-plus run pct: 11.3
15-plus mph pct: 20.6
We've spent enough time covering how Mack's broken hand played directly into Indianapolis' late-season descent. Without Mack, the Colts lacked the ball-carrier who broke 15 mph on 51 runs (fourth-most among running backs) and ripped off 28 runs of 10-plus yards. The team's most explosive runner will return in 2020 with the assistance of a similar back in rookie Jonathan Taylor. Multiply these numbers by two backs and consider the possibilities. It's not guaranteed, but the potential is enticing, and it shows how valuable Mack was to the Colts, even if he isn't exactly a household name.
Perhaps the most impressive part about Mack's breakout season is that he did it all despite missing 2 1/2 games with a broken hand, from which he was able to return before the initial prognosis.
Mack was the Colts' bell cow, reaching 20-plus touches in seven games, but he maintained his explosion without falling into methodical, chain-mover status.
His offensive coordinator, Nick Sirianni, certainly knows how valuable it's been to the Colts to have a capable No. 1 running back.
"Yeah, I think you have a good No. 1 back with Marlon that can do a lot of different things who is able to catch the ball out of the backfield, make explosive plays and make guys miss, get in the open field and open it up and pull away from the defense," Sirianni told reporters late last year when breaking down the Colts' backfield.
The third-year veteran finished ninth in the NFL by reaching at least 10 yards on 28 carries, was tied for third in runs of 20-plus yards and had one run of at least 40 yards (a 63-yard touchdown in Week 1 against the Log Angeles Chargers). He also had a pair of 10-plus-yard receptions.
Pro Football Focus has a metric called "Breakaway %," which measures how much of a runner's yardage is earned on big plays (runs of 15-plus yards). Mack ranked 10th in the NFL in 2019 with a 29.7-percent figure after totaling 13 such runs for 324 yards.
Another part of breaking long runs is avoiding tackles and being able to pick up yards after contact. Mack finished 12th in the NFL in yards after first contact with 693, also ranking 27th with an average of 2.81, and he was tied for 18th in avoided tackles with 39.
In total in 2019, Mack finished fifth in the league in first downs reached on runs (67), was tied for ninth in both carries (247) and rushing touchdowns (eight), was 11th in rushing yards (1,091) and tied for 23rd in yards per carry (4.4 avg.).
It was the 21st season in Colts franchise history in which a runner reached 1,000 yards, and Mack's total was the most by a Colts player since Edgerrin James' 1,506 rushing yards in 2005.
Colts' Dallis Flowers Named To PFWA All-Rookie Team
Flowers, who led the NFL in yards per kickoff return, was named to the PFWA's All-Rookie Team as a kick returner.
Colts 2023 Head Coach Search: Full List Of Interviews, Candidates
The Colts over the last few weeks have conducted several interviews for the team's head coaching position. Here's a recap of where things stand:
Colts 2023 NFL Mock Draft Monday: Jan. 23
Mock Draft Monday, our weekly roundup of experts' predictions for who the Colts will take in the NFL Draft, is back for 2023 this week with a focus on quarterbacks, as well as a few Day 2 selections. Check it out below.
Colts Interview New York Giants Defensive Coordinator Don 'Wink' Martindale For Head Coach Position
Martindale's aggressive defense helped propel the Giants to the playoffs in 2022.
Colts Interview New York Giants Offensive Coordinator Mike Kafka For Head Coach Position
Kafka spent five seasons coaching for Andy Reid with the Kansas City Chiefs before joining the Giants in 2022.
Colts Interview Dallas Cowboys Defensive Coordinator Dan Quinn For Head Coach Position
Quinn's Cowboys defense has helped Dallas reach the playoffs in each of the last two seasons.
Colts Interview Cincinnati Bengals Offensive Coordinator Brian Callahan For Head Coach Position
The Bengals' offense, under Callahan, has ranked seventh in points per game in each of the last two seasons.
Colts Interview Green Bay Packers Special Teams Coordinator Rich Bisaccia For Head Coach Position
Bisaccia has 21 years of experience as an NFL special teams coordinator.
Colts Interview Jeff Saturday For Head Coach Position
Saturday spent the final eight games of the 2022 season as the Colts' interim head coach.
2023 Colts Schedule Tracker: Live Updates
Keep track of all updates to the 2023 NFL schedule, including early announcements, international games & more!
Colts Interview Philadelphia Eagles Offensive Coordinator Shane Steichen For Head Coach Position
Steichen guided the Eagles to the NFL's second-highest points per game average in 2022.
Colts Interview Detroit Lions Defensive Coordinator Aaron Glenn For Head Coach Position
Glenn has spent the last two seasons with the Lions after serving as the New Orleans Saints' secondary coach from 2016-2020.


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Home News Candidates urged to back services
Candidates urged to back services
PLEDGE: Shoalhaven Neighbourhood Services administrator Maxine Edwards and Shoalhaven Women's Health Centre manager Tracy Lumb watch over South Coast Labor candidate Fiona Phillips and South Coast Greens candidate Amanda Findley as they sign a pledge to continue to support and strengthen funding into the community resources.
NOWRA'S community service workers are pleading with government candidates to protect and strengthen funding.
Shoalhaven Neighbourhood Services (SNS) and Shoalhaven Women's Health Centre (SWHC) workers spoke to local candidates on Thursday about funding concerns and the future of community services in the region.
South Coast Labor candidate Fiona Phillips and South Coast Greens Candidate Amanda Findley both signed a pledge to protect local community services in the lead-up to the election.SNS administrator Maxine Edwards said funds will end for the centre in June 2016, but would be part of competitive tenders to see the future of the company remain.
"We have been here for 37 years and during that time we haven't seen funding pressures to this level," she said."We are part of early intervention services.
Having local people within a local organisation means we are better suited to helping people and understanding what they can access in their area far easier than someone in Sydney.
"We're worried that the funding we are after will go towards crisis management centres rather than early intervention resources such as us that help strengthen and build our community.
"Ms Edwards said she had seen changes in other areas and was worried."Ulladulla lost four outreach programs in six months," she said.
"We believe funding contracts should be for five years so we have more stability.
"At the moment we have had to put time limitations on our staff contracts, which we have never had to do in the past. There's that insecurity with all our employees at the moment."
SWHC manager Tracy Lumb said scarce funds meant less staff and would make the tender for funds virtually impossible.
"We will need to go up against services like Mission Australia and the Salvation Army and so on for this funding and we don't even have a photocopier," Ms Lumb said."We are commercially disadvantaged before we even start.
"We also don't see huge donations like those organisations do. Last week I think we saw something like $7.80 and we obviously can't support our staff on that."
By JESSICA LONG


Wildlife watercolour illustration of a bat.
This British wildlife watercolour painting of a brown, long-eared bat was painted a couple of years ago and was inspired by forthcoming Halloween art activities.
The bat was quickly painted in a loose watercolour style and was a fun way to experiment with watercolours without the time required for a more detailed painting. An unexpected visit by a bat this morning just before sunrise, in the garden, was a timely reminder of Halloween and reminded me of this little bat watercolour.
Wildlife watercolour illustration of a bat. Wildlife Watercolours, brown long-eared bat.
The art of watercolour painting can include elements of fantasy and easily lends itself to children's book illustrations.
Read more about British bats in the wildlife garden blog.