Diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis in colon cancer cells

Cancer Letters 301 (2011) 193–202

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Diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis in colon cancer cells C. Lepage, D.Y. Léger, J. Bertrand, F. Martin, J.L. Beneytout, B. Liagre ⇑ Université de Limoges, Institut GEIST, EA 4021 «Biomolécules et thérapies anti-tumorales», Faculté de Pharmacie, Laboratoire de Biochimie, 87025 Limoges Cedex, France

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Article history: Received 20 October 2010 Received in revised form 8 December 2010 Accepted 8 December 2010

Keywords: Colon cancer cells Diosgenin Death receptor TRAIL p38 MAPK Apoptosis

a b s t r a c t Previously, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29. HT-29 cells have been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis. In this study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in HT-29 cells. We showed that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK pathway activation and subsequent DR5 overexpression. Furthermore, we showed that diosgenin alone, TRAIL alone or combination treatment increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Selectively inducing apoptosis in cancer cells has been increasingly recognized as a promising therapeutic approach for many cancers, including colorectal cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2 ligand) is a member of the tumor necrosis factor (TNF) cytokine family [1]. TRAIL is able to selectively induce apoptosis in tumor cells in vitro [1,2] and, most importantly in vivo [3,4] with no significant untoward effect on normal cells. Apoptosis induced by TRAIL is initiated by its binding to death receptors (TRAIL-R1 or DR4 and TRAIL-R2 or DR5) followed by formation of the death-inducing signalling complex (DISC) upon recruitment of specific cytoplasmic proteins, Fas-associated death domain (FADD) and Abbreviations: TRAIL, the tumor necrosis factor (TNF) a-related apoptosis-inducing ligand; DR, death receptor; MAPK, mitogen activated protein kinase; COX-2, cyclooxygenase-2. ⇑ Corresponding author. Tel./fax: +33 5 55 43 58 39. E-mail address: [email protected] (B. Liagre). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.12.003

caspase-8 or -10 [5]. Two other membrane bound receptors, DcR1 and DcR2, lack the cytoplasmic region and have truncated intracellular death domains, respectively, and cannot induce apoptosis. Although most cancer cells express functional TRAIL receptors DR4 and DR5, resistance to TRAIL’s cytotoxic effects is common. Many molecular mechanisms may account for these cancer cells’ resistance to TRAIL. Determinants of TRAIL sensitivity are reported to include the expression levels of death receptors (DR), FLICE inhibitor protein (FLIP) and Bcl-XL, but such relationships are not observed in all cell lines [6,7]. TRAIL sensitivity in tumor cell lines can be modulated by p53, MAP kinase pathways, c-Myc, PI3K/AKT pathway and NF-jB signalling [8,9]. Inhibition of PI3K/AKT activation or inhibition of NF-jB signalling by various agents has been shown to enhance TRAIL cytotoxicity in numerous cell models [10–12]. Thus, agents that can up-regulate TRAIL receptors or inhibit survival pathways, such as chemotherapeutic agents or natural products, have the potential to enhance the apoptotic effects of TRAIL [13–18].

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Diosgenin, a steroid saponin present in fenugreek (Trigonella foenum graecum) and other plants, is known for its antitumor effects on cancer cells [19]. Diosgenin inhibits the growth of cancer cell lines through cell cycle arrest, inhibition of PI3K/Akt and NF-jB survival pathways and induction of apoptosis [20–22]. In our previous study, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29 [23]. After diosgenin treatment, we observed apoptosis hallmarks in both cell lines but HT-29 cells were more resistant with delayed apoptosis. On the other hand, HT-29 cells have also been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis [24]. Therefore, a combinatorial strategy using diosgenin seemed to be potentially promising for overcoming TRAIL resistance in colorectal cancer cells. Cyclooxygenase-2 (COX-2) expression is usually involved in colorectal cancer pathogenesis [25,26]. Tang et al. [27] showed that COX-2 overexpression inhibited DR5 expression and attenuated TRAIL-induced apoptosis in human colon cancer cells and this inhibition was restored by the COX-2 inhibitor sulindac. The causal relationship between arachidonic acid metabolism and apoptosis induced by TRAIL must be probed to establish whether it is a well-founded target in colorectal cancer treatment. In the current study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in human colon cancer cells HT-29. We showed for the first time that diosgenin sensitizes HT-29 colon cancer cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK signalling pathway activation and subsequent DR5 overexpression in HT-29 cells. Furthermore, diosgenin also activated apoptotic effectors and inhibited survival pathways thus amplifying TRAIL-induced apoptosis. On the other hand, we showed that diosgenin alone, TRAIL alone or a combination of both increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction. Taken together, these results suggest that the combination of diosgenin with TRAIL may be a promising candidate for treatment of TRAIL-resistant colon cancer cells.

2. Materials and methods 2.1. Cell lines, cell culture, treatment and light microscopy The HT-29 cell line was purchased from American Culture Type Collection (LGC Standards, Middlesex, United Kingdom). Cells were seeded at 5  106 cells in 75 cm2 tissue culture flasks, grown in DMEM medium (Gibco BRL, Cergy-Pontoise, France) supplemented with 5% fetal calf serum (FCS) (Gibco BRL), 100 U/ml penicillin and 100 lg/ ml streptomycin (Gibco BRL). The HCT-116 cell line was purchased from American Culture Type Collection (LGC Standards). Cells were seeded at 3  106 cells in 75 cm2 tissue culture flasks, grown in MEM medium (Gibco BRL) supplemented with 5% fetal calf serum (FCS) (Gibco BRL), 100 U/ml penicillin and 100 lg/ml streptomycin (Gibco BRL). The HEL human erythroleukemia cell line was kindly provided by Pr JP. Cartron (INSERM U76, Paris, France).

Cells were seeded at 105 cells/ml in 75 cm2 tissue culture flasks and grown in RPMI 1640 medium (Gibco BRL) as previously described [28,29]. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Cells were grown for 36 h in culture medium prior to exposure or not to 20 or 40 lM diosgenin (Sigma Aldrich, Saint Quentin Fallavier, France) 6 h before adding or not 20 ng/mL TRAIL (R&D System, Lille, France). A stock solution of 10 2 M diosgenin was prepared in ethanol; a 20 lg/mL stock solution of TRAIL was prepared in phosphate-buffered saline (PBS) – 0.1% BSA, stock solutions were then diluted in culture medium to give the appropriate final concentration. The same amount of vehicle was added to control cells. Cell viability was determined by the trypan blue dye exclusion method. For light microscopy, after treatment, cultured cells were examined under phase-contrast microscopy (magnification 400), and pictures were taken with an image acquisition system (Nikon, Champigny sur Marne, France). When the pharmacological inhibitor of COX-2, celecoxib, was used, cells were treated or not simultaneously with 10 lM celecoxib and 20 or 40 lM diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h. For p38 inhibition, cells were pretreated with 10 lM SB203580 (Calbiochem, La Jolla, CA, USA) for 2 h and then treated or not with 40 lM diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h. 2.2. Apoptosis quantification: DNA fragmentation HT-29 cells were seeded at 5  106 cells in 75 cm2 tissue culture flasks and then treated as described above. Apoptosis was quantified on floating and adherent cells using ‘‘cell death’’ enzyme-linked immunosorbent assay (ELISA) (Cell Death Detection ELISAPLUS, Roche Diagnostics). Cytosol extracts were obtained according to the manufacturer’s protocol and apoptosis was measured as previously described [20]. 2.3. Protein expression analysis After treatment, cells were washed and lysed in RIPA lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.1% SDS, 20 lg/ml aprotinin) containing protease inhibitors (Complete™ Mini, Roche Diagnostics, Meylan, France). Western blot was performed as previously described [30]. Briefly, proteins (10–100 lg) were separated by electrophoresis on SDS–polyacrylamide gels, transferred to PVDF membranes (Amersham Pharmacia Biotech, Saclay, France) and probed with respective antibodies against poly(ADP-ribose) polymerase (PARP), procaspase-3, phospho-Akt, phospho-p38, DR4 and DR5 (Santa Cruz Biotechnology; TEBU, Le Perray en Yvelines, France), against caspase-8, caspase-9 and Bid (Cell Signalling Technology, Ozyme) and against COX-2 (Cayman Chemical). After incubation with secondary antibodies (Dako), blots were developed using enhanced chemiluminescence reagents (Amersham). Membranes were then reblotted with anti-b-actin monoclonal antibody (Sigma). Western blots were analyzed by densitometry (GBOX,

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Procaspase-8 Cleaved caspase-8 Procaspase-9 Cleaved caspase-9 Bid Cleaved Bid Procaspase-3 PARP Cleaved PARP β-actin Fig. 1. Diosgenin enhanced TRAIL-induced apoptosis. (A) HT-29 cells were treated or not with 20 or 40 lM (Dios20 and Dios40) diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h. The cell morphology was visualized with phase-contrast microscopy (magnification, 400). (B) Diosgenin plus TRAIL induced sub-G1 cell population in HT-29 cells. DNA contents were analyzed by flow cytometry analysis. (C) Diosgenin caused a significant increase of TRAIL-induced DNA fragmentation. Apoptosis was quantified on floating and adherent cells using ‘‘cell death’’ ELISA based on DNA fragmentation. The fold induction (apoptotic ratio) of DNA fragmentation is normalized relative to the value of control culture, which is taken as 1. Data are expressed as mean ± SD of three independent experiments. ⁄p < 0.05 or #p < 0.05 (Fisher’s PLSD test) is considered to indicate significance versus untreated cells group or TRAIL group respectively. (D) Expression of apoptosis markers after combined treatment with diosgenin and TRAIL. Protein extracts prepared from the cells were subjected to Western blotting analysis using specific antibodies to caspase-8, caspase-9, Bid, procaspase-3, PARP and b-actin. One representative of three independent experiments is shown.

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Ozyme, France) and protein expression was normalized to b-actin. 2.4. Evaluation of cell phenotype and fluorescence-activated cell sorting for death receptors For DNA content analysis, cells were treated or not and then harvested, fixed and permeabilized in 70% ethanol in PBS at 20 °C overnight, washed in PBS, treated with RNase (40 U/lL, Boehringer Mannheim, Meylan, France) for 20 min at room temperature, and stained with propidium iodide (PI) (50 lg/ml). Flow cytometric analyses were performed as described previously [31]. 2.5. Assay of PGE2 production

binds with phosphorylated and non-phosphorylated protein. After washes and incubation with blocking buffer, 100 ll of phospho-MAPK standard or diluted samples were loaded in each well and incubated 2 h at room temperature. After washing, a biotinylated detection antibody specific for phospho-ERK1/ERK2, phospho-JNK or phosphop38 was used to detect only phosphorylated protein. After 2 h incubation, standard streptavidin-HRP was added to reveal phospho-MAPKs during 20 min. After washing, 100 ll substrate solution were incubated for 20 min and stop solution was added before reading the microplate at 450 nm. Quantitative sample values of MAPK phosphorylated forms were determined after generation of a standard curve and normalization with cell number. 2.7. Statistical analysis

PGE2 levels in culture medium were measured by enzyme immunoassay. Measurements were quantified on supernatants from treated or control HT-29 cells. The PGE2 concentration in the medium was measured using an ELISA kit according to the manufacturer’s instructions (Cayman Chemical) and was normalized with respect to the number of viable cells present in the particular culture at the time of sampling.

The medians and standard deviations (SD) were calculated using Excel (Microsoft Office, Version 98). Statistical analysis of differences was carried out by analysis of variance (ANOVA) using StatView Version 5.0 (SAS Institute Inc, Cary, North Carolina). A P-value less than 0.05 (Fisher’s Protected-least-significant-difference (PLSD) test) was considered to indicate significance. 3. Results

2.6. Quantification of MAP kinase activation by ELISA After treatment, 106 cells were homogenized in lysis buffer containing protease and phosphatase inhibitors in accordance with the manufacturer’s protocol (R&D Systems, Lille, France) as previously described [32]. Before assay, the microplate was coated with capture antibody (4 lg/ml) overnight at room temperature. This antibody

3.1. Diosgenin enhances TRAIL-induced apoptosis in HT-29 cells To determine whether diosgenin could enhance TRAIL-induced apoptosis in HT-29 colon cancer cells. Cells were pretreated with diosgenin (20 lM Dios20 or 40 lM Dios40) for 6 h, and then exposed or not to TRAIL (20 ng/mL) for 24 h. Direct observation with phase-contrast microscopy demonstrated that diosgenin alone did not cause morphological modifications or cell death regardless of the dose used (Fig. 1A). On the other

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Fig. 2. Diosgenin induced DR5 expression in a dose- and time-dependent manner. (A) HT-29 cells were treated or not with 10, 20 or 40 lM diosgenin for 24 h. (B) HT-29 cells were treated or not with 40 lM (Dios40) diosgenin for 6, 12 or 24 h. (C) HCT116 and HEL cells were treated or not with 40 lM diosgenin for 24 h. For all experiments, protein extracts were subjected to Western blotting analysis using specific antibodies to DR4, DR5 and b-actin. For each western blot, one representative of three independent experiments is shown.

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and a slight activation of caspase-9 and bid (Fig. 1D). The combined treatment of HT-29 cells with diosgenin and TRAIL resulted in an increase in caspase-8 and -9 activation and elevated caspase-3 activation and PARP cleavage in a diosgenin dose-dependent manner (Fig. 1D). Taken together, our results indicate that diosgenin can enhance TRAIL-induced apoptosis.

3.2. Diosgenin upregulates DR5 expression in HT-29 cells To further understand the mechanism by which diosgenin potentiates TRAIL-induced apoptosis in HT-29 cells, its effect on death receptors DR4, DR5, DcR1 and DcR2 expressions were studied. For this, HT-29 cells were treated with different concentrations of diosgenin for 24 h, and death receptor expressions were evaluated. Untreated HT-29 cells expressed the four death receptors at different levels with basal expression of DR4 and DR5 and weak expression of DcR1 and DcR2 (data not shown). Diosgenin treatment had no effect on DR4 expression (Fig. 2A, left) but strongly increased DR5 expression (Fig. 2A, right) in a dose-dependent manner. We also examined whether the diosgenin effect was dependent on time and we showed that in HT-29 cells treated by 40 lM diosgenin, DR5 expression increased in a time-dependent manner (Fig. 2B, right) and that DR4 expression was constant regardless of the duration of treatment (Fig. 2B, left). The expression of DcR1 and DcR2 receptors was not induced by diosgenin treatment (data not shown).

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hand, a few cells underwent morphological modifications after treatment with TRAIL alone. The rate of cells undergoing morphological modifications increased when TRAIL was used in combination with diosgenin and this, in a dose-dependent manner (Fig. 1A). We then determined whether these treatments induced apoptosis in HT-29 cells. Cells undergoing apoptosis will lose part of their DNA due to DNA fragmentation in late stage apoptosis. These cells may be detected as a sub-G1 population by flow cytometry. Furthermore, quantitative determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) induced during apoptosis was done by enzyme immunoassay. After 24 h diosgenin treatment, we showed the absence of a sub-G1 peak and of DNA fragmentation (Fig. 1B and C). These data were consistent with our previous study [23]. As for TRAIL alone treatment, we observed a slight increase in the sub-G1 population (+3.9% versus untreated cells, p < 0.05) and DNA fragmentation (+4.6-fold versus untreated cells, p < 0.05). Interestingly, combined treatment of TRAIL with 20 or 40 lM diosgenin enhanced the sub-G1 peak (+7.6% and +16.6% respectively versus TRAIL alone, Fig. 1B) and DNA fragmentation (+1.8- and +3.1-fold respectively versus TRAIL alone, Fig. 1C) in a dose-dependent manner. Next, we examined the effect of diosgenin, TRAIL, and the combination on activation of apoptosis effectors such as caspase-8, caspase-9, bid, procaspase-3, or PARP cleavage. We found that diosgenin alone for 24 h did not activate apoptosis effectors (Fig. 1D). As for TRAIL alone, we observed moderate activation of caspase-8 and caspase-3 (as shown by decreased procaspase-3 levels) followed by subsequent PARP cleavage

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Fig. 3. p38 activation and Akt downregulation were implicated in diosgenin sensitization to TRAIL-induced apoptosis. HT-29 cells were treated with 40 lM (Dios40) diosgenin for 6 h or 20 ng/mL TRAIL for 24 h or combined treatment. Quantitative phosphorylated levels of MAPK were determined by ELISA (upper panel and lower left panel). Data are expressed as mean ± SD of three independent experiments. ⁄p < 0.05 (Fisher’s PLSD test) is considered to indicate significance in comparison to untreated cells group. Protein extracts were subjected to Western blotting analysis using specific antibodies to Phospho-Akt and b-actin. One representative of three independent experiments is shown (lower right panel).

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C. Lepage et al. / Cancer Letters 301 (2011) 193–202 HT-29 cells were treated with 40 lM diosgenin for 6 h or TRAIL for 24 h or combined treatment. Quantitative phosphorylated levels of MAP kinases were determined by ELISA (Fig. 3 upper panel and lower left panel). We found that p38 was the only MAPK for which phosphorylation was increased by diosgenin treatment (+1.9-fold versus untreated cells, p < 0.05, Fig. 3 lower left panel). On the other hand, it seemed that TRAIL treatment had no particular effect on MAPK phosphorylation and that combined treatment (+1.8-fold versus untreated cells) had no further effect in comparison with diosgenin alone. Activation of the Akt survival pathway can contribute to TRAIL resistance in cancer cells [34]. Interestingly, the level of phospho-Akt protein was decreased after diosgenin treatment versus untreated cells whereas TRAIL alone had no effect and combined treatment had no further effect (Fig. 3 lower right panel). Thus, our results suggest that p38 MAPK signalling and inactivation of the Akt survival pathway by diosgenin play a part in sensitization to TRAIL-induced apoptosis.

We also investigated whether the diosgenin effect on death receptors was specific to HT-29 or also occured in other cell types. For this, another colon cancer cell line (HCT-116), and erythroleukemia cells (HEL) were exposed to 40 lM diosgenin for 24 h and then examined DR4 and DR5 protein expression. As observed in HT-29 cells, diosgenin had no effect on DR4 expression level in HCT-116 or HEL cells and increased DR5 expression in the two cell lines tested (Fig. 2C). These findings suggest that the up-regulation of DR5 by diosgenin is not cell type specific. Taken together, these results suggest that diosgenin can sensitize HT29 cells to TRAIL-induced apoptosis in part through upregulation of DR5 expression.

3.3. Diosgenin induces p38 MAPK signalling and downregulation of Akt survival pathway in HT-29 cells MAP kinases and PI3K/Akt signalling pathways are well known regulators of chemically-induced apoptosis that are activated upon phosphorylation. Among MAP kinases, ERK1/2 are preferentially activated by growth factors, whereas JNK and the p38 MAPKs are preferentially activated by cell stress-inducing signals, such as oxidative stress, environmental stress, and pro-apoptotic treatment [33]. Thus, we investigated the effects of diosgenin alone, TRAIL alone or combined treatment on the activity of MAPK kinases in order to determine whether this signalling pathway mediated the observed apoptotic response. In this experiment,

3.4. DR5 up-regulation and enhanced sensitivity to TRAIL induced by diosgenin are reversed by p38 MAPK inhibitor As diosgenin activated p38 MAPK after 6 h treatment we evaluated the kinetic of p38 activation after diosgenin treatment. HT-29 cells were then treated by 40 lM diosgenin for 2, 4, 6, 12 and 24 h and phospho-p38 levels were assessed by western-blotting. We found that diosgenin

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Fig. 4. Selective inhibition of p38 pathway decreased diosgenin-induced DR5 up-regulation and blocked diosgenin sensitization to TRAIL-induced apoptosis. (A) HT-29 cells were treated by 40 lM diosgenin for 2, 4, 6, 12 and 24 h and phospho-p38 levels were assessed by western-blotting. One representative of three independent experiments is shown. (B) HT-29 cells were pre-incubated or not with 10 lM SB203580 for 2 h and treated with 40 lM (Dios40) diosgenin for 6, 12 and 24 h. Protein extracts prepared from the cells were subjected to Western blotting analysis using specific antibodies to Phospho-p38, DR5 and b-actin. One representative of three independent experiments is shown. (C) HT-29 cells were pre-incubated or not with 10 lM SB203580 for 2 h, then treated or not with 40 lM (Dios40) diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h. Apoptosis was quantified on floating and adherent cells using ‘‘cell death’’ ELISA based on DNA fragmentation. The fold induction (apoptotic ratio) of DNA fragmentation is normalized relative to the value of the control culture, which is taken as 1. Data are expressed as mean ± SD of three independent experiments. ⁄p < 0.05 (Fisher’s PLSD test) is considered to indicate significance in comparison to Dios40/TRAIL group. ns = non-significant compared to TRAIL group.

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3.5. COX-2 involvement in apoptosis induced by diosgenin, TRAIL and combined treatment Previously, we showed that COX-2 was expressed in HT-29 cells [35] and was implicated in diosgenin-induced apoptosis in these cells [23]. In fact, we demonstrated that colorectal cancer cells expressing COX-2 in basal conditions were more resistant than COX-2 deficient cells [23]. In different cell lines, COX-2 expression and activity were increased after diosgenin treatment [23,30,31]. These results suggest that COX-2 may act as a survival pathway activated during diosgenin treatment. COX-2 activity is notably mediated by the prostaglandin E2 (PGE2), its main metabolite. We therefore examined the expression of COX-2 by Western blot analysis and its activity by PGE2 ELISA assay in HT-29 cells. We found that diosgenin enhanced the expression and activity of COX-2 in a dosedependent manner (Fig. 5A and B). Interestingly, TRAIL had the same effect on COX-2 expression and activity and combined treatment with

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induced sustained p38 activation that peaked after 6 and 24 h treatment (Fig. 4A). As diosgenin, and to a lesser extent TRAIL, activated p38 MAPK, we studied whether the apoptosis caused by the combination of diosgenin and TRAIL could be rescued by SB203580, a specific p38 inhibitor. HT-29 cells were pre-incubated with 10 lM SB203580 for 2 h, then treated with diosgenin, or TRAIL or a combination of both. Then, DR5 protein expression and apoptosis were evaluated. We found that pretreatment of HT-29 cells by SB203580 not only blocked diosgenin-induced p38 activation but also abolished diosgenin-induced up-regulation of DR5 (Fig. 4B). Furthermore, we demonstrated that SB203580 pretreatment slightly reduced TRAIL-induced apoptosis but especially suppressed TRAIL sensitization induced by diosgenin during the combination treatment (Fig. 4C). Thus, these experiments confirm that diosgenin sensitizes HT29 cells to TRAIL through notably involvement of p38 phosphorylation.

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Fig. 6. Celecoxib decreased PGE2 synthesis induced by diosgenin and TRAIL combined treatment. (A) HT-29 cells were treated or not simultaneously with 10 lM celecoxib and 20 or 40 lM (Dios20 and Dios40) diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h and COX-2 levels were assessed by western-blotting. One representative of three independent experiments is shown. (B) PGE2 levels in culture medium were measured by enzyme immunoassay. Measurements were quantified on supernatants from treated or not HT-29 cells. Data are expressed as mean ± SD of three independent experiments. ⁄p < 0.05 (Fisher’s PLSD test) is considered to indicate significance versus Dios20/TRAIL group or Dios40/TRAIL group respectively.

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Fig. 5. Effect of diosgenin and TRAIL combined treatment on COX-2 expression and activity. (A) HT-29 cells were treated or not with 20 or 40 lM (Dios20 and Dios40) diosgenin for 6 h before adding or not 20 ng/ mL TRAIL for 24 h and COX-2 levels were assessed by western-blotting. One representative of five independent experiments is shown. (B) PGE2 levels in culture medium were measured by enzyme immunoassay. Measurements were quantified on supernatants from treated or not HT29 cells. Data are expressed as mean ± SD of five independent experiments. ⁄p < 0.05 or #p < 0.05 (Fisher’s PLSD test) is considered to indicate significance versus untreated cells group or TRAIL group respectively.

diosgenin increased even more intensely its expression and activity (Fig. 5A and B) and still in a dose-dependent manner, especially at 40 lM diosgenin (+2.6-fold versus TRAIL alone, p < 0.05, Fig. 5B). Thus, to examine the relationship between COX-2 and induced apoptosis, we used celecoxib, a specific inhibitor of COX-2 activity. HT-29 cells were treated simultaneously with 10 lM celecoxib and 20 or 40 lM diosgenin for 6 h before adding TRAIL for 24 h. We showed that celecoxib did not modify COX-2 up-regulation levels (Fig. 6A) but induced a strong decrease in PGE2 release induced by combined treatment ( 1.4-fold Dios20 + TRAIL and 2-fold Dios40 + TRAIL, p < 0.05, Fig. 6B). We therefore evaluated apoptotic features of HT-29 cells treated by celecoxib and combined diosgenin + TRAIL treatment. For this, we evaluated DR5 expression and quantified DNA fragmentation after treatments. As found in other studies [36,37], we found that celecoxib alone increased DR5 expression but had no apoptotic effect per se (Fig. 7A and B). In fact, celecoxib was able to enhance DR5 expression (Fig. 7A) and apoptosis induced by combined treatment (+1.5-fold versus combined treatment, p < 0.05, Fig. 7B) and thus further sensitized HT-29 cells to TRAIL-induced apoptosis.

4. Discussion Use of natural agents and selectively inducing apoptosis in colon cancer cell are promising therapeutic approach. Among all the apoptosis-inducing cytokines, TRAIL is the only cytokine that can selectively induce apoptosis in tumor cells without affecting normal cells [4]. But, resistance of cancer cells to TRAIL is one of the major roadblocks to the development of this therapy [7]. Thus a combination

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Fig. 7. Celecoxib increased DR5 expression and DNA fragmentation caused by diosgenin and TRAIL combined treatment. (A) HT-29 cells were treated or not simultaneously with 10 lM celecoxib and 40 lM (Dios40) diosgenin for 6 h before adding or not 20 ng/mL TRAIL for 24 h and DR5 levels were assessed by western-blotting. One representative of three independent experiments is shown. (B) Apoptosis was quantified on floating and adherent cells using ‘‘cell death’’ ELISA based on DNA fragmentation. The fold induction (apoptotic ratio) of DNA fragmentation is normalized relative to the value of the control culture, which is taken as 1. Data are expressed as mean ± SD of three independent experiments. ⁄ p < 0.05 (Fisher’s PLSD test) is considered to indicate significance in comparison to Dios40/TRAIL group.

of TRAIL with another agent to overcome this resistance paves the way for new anticancer therapy [13–18,38]. In the present study, we studied the potential sensitizing effect of diosgenin on TRAIL in HT-29 colon cancer cells. Previous studies showed that diosgenin induced apoptosis in various cancer cells including HCT-116 and HT-29 colon cancer cells [20–23,39–41] but HT-29 cells were more resistant than others. On the other hand, HT-29 cells have also been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis [24]. Therefore, it was interesting to investigate the combination of these two anticancer agents. Interestingly, we showed for the first time that the combined treatment significantly increased TRAIL-induced apoptosis in a diosgenin dosedependent manner. We also found that the mechanism by which diosgenin mediated its effects on TRAIL-induced apoptosis appears to involved the induction of TRAIL death receptor DR5 and inhibition of the PI3K/Akt survival pathway. We showed that DR5 induction by diosgenin was not cell type specific but was observed in another colon cancer cell lines HCT116 and erythroleukemia cells HEL. Furthermore, the combination of diosgenin with TRAIL amplified TRAIL-induced activation of apoptosis effectors and particularly activation

of caspase-8 and the executioner caspase-3 leading to increased PARP cleavage and DNA fragmentation. These results imply that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis in part through up-regulation of DR5 cell expression leading to subsequent amplification of TRAIL cell death signals. There are many factors contributing to resistance to TRAIL-induced apoptosis. Among the cell signalling pathways that promote cell survival, Akt is one of the most important [34,42]. Diosgenin is known to suppress Akt activation in various cell types [21,22,43]. In contrast, TRAIL can activate the Akt pathway and blockade of Akt phosphorylation sensitizes resistant cells to TRAIL [34,44]. In our model, we demonstrated that diosgenin alone decreased Akt phosphorylation in HT-29 cells. With combined treatment, we showed that Akt activation was also decreased. Thus, diosgenin is able to abrogate the Akt survival pathway normally started by TRAIL and so sensitizes HT-29 cells to TRAIL-induced apoptosis. MAPKs play a central role in transduction of signal of growth and differentiation and also act as important modulators of various apoptosis-inducing signals [33]. It is known that the dynamic balance between growth factoractivated ERK and stress-activated JNK and p38 pathways determine whether a cell survives or undergoes apoptosis [33]. But, in TRAIL-induced apoptosis, the role of MAPK is controversial [45]. In a previous study, we showed that diosgenin can induce apoptosis with p38 activation [46]. In this report, we showed that in HT-29 cells, diosgenin induced a sustained activation of the p38 MAPK pathway but did not affect ERK or JNK pathways. We also found that sensitization of HT-29 cells to TRAIL and DR5 up-regulation induced by diosgenin required p38 MAPK activation. Selective inhibition of p38 activation by SB203580, blocked diosgenin-induced DR5 up-regulation and abolished diosgenin-induced sensitization of HT-29 cells toward TRAIL. Other studies have also demonstrated that p38 activation led to the up-regulation of TRAIL death receptors DR4 and DR5 and was involved in the sensitization effect of various natural products and chemotherapeutic agents [47–50]. Thus, we confirmed the involvement of p38 in diosgenin sensitization of HT-29 cells to TRAIL. We previously described the involvement of COX-2 in diosgenin-induced apoptosis in osteosarcoma cells [31] and recently in HT-29 colorectal cancer cells [23]. In this last study, diosgenin induced delayed apoptosis with increased COX-2 expression and activity. In the presence of NS-398, a specific inhibitor of COX-2 expression, we observed an increase in apoptosis rate allowing us to conclude that COX-2 played a protective role against diosgenin. Moreover, Tang et al. [27] showed that COX-2 overexpression protects colon cancer cells from TRAIL-induced apoptosis. In the present study, we confirmed that diosgenin increased COX-2 expression and activity in a dose-dependent manner and showed that TRAIL exerted the same effect on this enzyme. We verified the potential pro-survival effect of COX-2 induction after diosgenin and/or TRAIL treatment using celecoxib, a specific inhibitor of COX-2 activity. Different studies demonstrated that COX-2 inhibition can sensitize cancer cells to TRAIL [51,52] and celecoxib is known to sensitize cancer cells

C. Lepage et al. / Cancer Letters 301 (2011) 193–202

SB203580

Dios

CX

PGE2 +

Apoptosis effectors +

Akt -

p38 ++

DR5 ++

APOPTOSIS +

CX

SB203580

TRAIL

Apoptosis effectors +

PGE2 +

p38 +

APOPTOSIS +

SB203580

Dios

CX

TRAIL PGE2 ++

p38 ++

Akt -

DR5 ++

Apoptosis effectors ++

APOPTOSIS +++ Fig. 8. Schematic summary of signalling pathways involved in diosgenin sensitization to TRAIL-induced apoptosis associated with p38 activation, DR5 up-regulation and Akt downregulation in human HT-29 cells in vitro.

to TRAIL-induced apoptosis [36,37]. In our model, besides decreasing PGE2 release, celecoxib up-regulated DR5 and further enhanced apoptosis induced by diosgenin and TRAIL combined treatment so confirming the protective role of COX-2 in our model. Overall, we conclude, for the first time, that diosgenin can enhance TRAIL-induced apoptosis through the activation of the p38 MAPK pathway, subsequent DR5 up-regulation and downregulation of the PI3K/Akt survival pathway (Fig. 8). Considering that diosgenin when used alone has been demonstrated to be safe and exhibits antitumor effects in vitro [20,21,23,40,41] and in vivo [39,43] against a wide variety of tumors, it could be used in combination with TRAIL particularly in cases of tumors that develop resistance to TRAIL. Conflicts of interest No potential conflicts of interest were disclosed. Acknowledgments We acknowledge Dr. J. Cook-Moreau for corrections in the preparation of this manuscript. This work was supported in part by the Ministère de l’Education Nationale, de la Recherche et de la Technologie, the Conseil Régional

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