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Endocrinology, doi:10.1210/en.2008-0275
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Endocrinology Vol. 149, No. 8 3789-3798
Copyright © 2008 by The Endocrine Society

X-Linked Inhibitor of Apoptosis Protein Levels and Protein Kinase C Activity Regulate the Sensitivity of Human Endometrial Carcinoma Cells to Tumor Necrosis Factor{alpha}-Induced Apoptosis

Céline Van Themsche, Lyne Lafontaine and Eric Asselin

Research Group in Molecular Oncology and Endocrinology, Department of Chemistry and Biology, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada G9A 5H7

Address all correspondence and requests for reprints to: Dr. Eric Asselin, Université du Québec à Trois-Rivières, 3351 Boulevard des Forges, Casier Postal 500, Trois-Rivières, Québec, Canada G9A 5H7. E-mail: eric.asselin{at}uqtr.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endometrial carcinomas are often chemoresistant. TNF{alpha} shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNF{alpha} to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNF{alpha} triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNF{alpha} up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNF{alpha}-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNF{alpha}-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNF{alpha}-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNF{alpha}-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNF{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL CARCINOMA is the leading type of gynecological cancer in the Western world. Endometrial tumors often show resistance to current chemotherapeutic drugs (1, 2), and battle against this cancer would benefit from the development of alternative therapeutic approaches. TNF{alpha} is a proinflammatory cytokine that can induce apoptosis in a variety of cancer cell types (3). The cytokine can bind and activate two receptors, TNF-R1 and TNF-R2 (4). Although it has been reported that both receptors are required for the induction of apoptosis by TNF{alpha} (5), activation of caspase signaling leading to apoptotic cell death is generally mediated by TNF-R1 (6). Upon TNF{alpha} binding, activation of TNF-R1 induces the recruitment of adaptor molecules and procaspase-8 to the receptor complex (7), thereby initiating a cascade of caspase activation (involving caspase-9 and -3) and proteolytic degradation of key repair and housekeeping proteins, such as poly(ADP-ribose) polymerase (PARP) (8). TNF{alpha}-induced activation of MAPK pathway MAPK kinase (MEK) /ERK and particular protein kinase C (PKC) isoforms enhances the proapoptotic effect of TNF{alpha} (9), but TNF{alpha} can also activate an antiapoptotic pathway involving phosphatidylinositide 3-kinase (PI3-K)/Akt, nuclear factor-{kappa}B (NF-{kappa}B) and particular PKC isoforms, which can protect cancer cells from TNF{alpha}-induced apoptosis (10).

The antitumor effects of TNF{alpha} have been described in detail for particular cancers and are now exploited in anti-cancer trials (reviewed in Ref. 11). These trials involve local delivery of TNF{alpha} because systemic administration of the proinflammatory cytokine at high doses can cause a severe condition called septic shock (12). Endometrial tumors are generally restricted to the uterus and could be targeted locally using noninvasive treatments such as the use of pump devices. Because encouraging studies have reported that TNF{alpha} can inhibit the growth of various endometrial carcinoma cell lines (13, 14), we hypothesized that TNF{alpha} could efficiently induce apoptosis in endometrial carcinoma cells. In this study, we have thus tested the antitumor activity of TNF{alpha} in endometrial carcinoma cells.

In addition, the identification of factors regulating cellular sensitivity to TNF{alpha} may provide new therapeutic targets to enhance the proapoptotic activity of the cytokine in endometrial carcinoma cells. In this regard, we have gathered evidence of a key antiapoptotic role for X-linked inhibitor of apoptosis protein (XIAP) in endometrial carcinoma cells. This member of the inhibitor of apoptosis protein family can directly inhibit caspases-3, -7, and -9 (15), and we recently observed that XIAP protects endometrial carcinoma cells against various proapoptotic agents, including TGF-β (16) and chemotherapeutic drugs (17). Because XIAP has been reported to confer resistance to TNF{alpha}-induced apoptosis in non-cancer cells (18), we hypothesized that XIAP also protects endometrial carcinoma cells against TNF{alpha}-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents
Endometrial carcinoma Ishikawa and Hec-1A cell lines were purchased from ATCC (www.atcc.org). The two cell lines were derived from two different patients; Ishikawa cells were obtained from a well-differentiated endometrial carcinoma, whereas Hec-1A cells were derived from a poorly differentiated endometrial carcinoma. Ishikawa cells were maintained in DMEM-F12 medium containing 15 mM HEPES, 2% bovine growth serum, and 50 mg/ml gentamycin. Hec-1A cells were maintained in McCoy’s medium supplemented with 5% BGS and 50 mg/ml gentamycin. All antibodies were purchased from Cell Signaling Technology (Beverly, MA) [P-Akt (60 kDa), total Akt (60 kDa), XIAP (53 kDa), cleaved caspase-8 (10 kDa), cleaved caspase-9 (35 kDa), cleaved caspase-3 (19, 17, 12 kDa), cleaved PARP (89 kDa), phospho-PKC (P-PKC) (85 kDa), P-P44/42 (44,42 kDa)], except for horseradish peroxidase-conjugated goat antirabbit secondary antibody (Bio-Rad Laboratories, Mississauga, Ontario, Canada), antihuman TNF-R1 antibody (RandD Systems, Minneapolis, MN) and CY3-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). Recombinant TNF{alpha} was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). PI3-kinase inhibitor LY294002 and MEK1 inhibitor PD98059 were purchased from Cell Signaling Technology. PKC inhibitors chelerythrine chloride (which inhibits PKC {alpha}, β, {delta}, and {gamma}) and calphostin C, which inhibits PKC {eta} and {theta} in addition to PKC {alpha}, β, {delta}, {epsilon}, and {gamma}, were obtained from Sigma. XIAP small interfering RNA (siRNA) was obtained from Invitrogen (Burlington, Ontario, Canada).

RNA isolation and RT-PCR
Total RNA was isolated from cells using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 0.4 µg RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Primers for amplification of TNF-R1 were 5'-ACCAAGTGCCACAAAGGAAC-3' (S) and 5'-CTGCAATTGAAGCACTGGAA-3' [antisense (AS)]; primers for TNF-R2 were 5'-TTCGCTCTTCCAGTTGGACT-3' [sense (S)] and 5'-CACCAGGGGAAGAATCTGAG-3' (AS); primers for XIAP were 5'-GAGAAGATGACTTTTAACAGTTTTGA-3' (S) and 5'-TTTTTTGCTTGAAAGTAATGACTGTGT-3' (AS); primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-GTCAGTGGTGGACCTGACCT-3' (S) and 5'-TGAGCTTGACAAAGTGGTCG-3' (AS). PCRs were conducted in a MJ Research (Ramsey, MI) Thermal cycler (model PTC-100), using the following parameters: 30 sec at 94 C, 30 sec at 58 C, and 1 min at 72 C for 35 cycles, except for GAPDH (25 cycles). The reaction mixture was size-separated on an agarose gel and visualized using SYBR-Safe (Invitrogen) staining upon UV transillumination.

Monitoring of TNF-R1 expression using flow cytometry
Ishikawa and Hec-1A cells were trypsinized and fixed in 2% formalin for 1 h. No permeabilization was performed to ensure exclusive labeling of cell surface proteins. Cells were labeled for 2 h with mouse antihuman TNF-R1 antibody diluted to 2.5 µg/ml in PBS-0.5% BSA (control cells were labeled with 2% bovine serum), incubated with CY3-conjugated goat antimouse secondary antibody in PBS-0.5% BSA for 30 min and analyzed with Cytomics FC 500 flow cytometer (Beckman-Coulter, Mississauga, Ontario, Canada) using FL2 filter (575 nm).

Hoechst nuclear staining
Treated cells were collected and resuspended in 1 µg/ml Hoechst 33258 (Sigma) in 4% formalin and incubated for 24 h at 4 C before blind cell counts were performed. At least 200 cells were counted for each sample, and the percentage of apoptotic cells was calculated as the ratio of apoptotic cells (with characteristic apoptotic morphology such as nuclear shrinkage and condensation, as revealed by fluorescence microscopy) to total cell count.

Terminal deoxynucleotidyl transferase deoxyuridine triphosphosphate nick-end labeling (TUNEL) assay
Treated cells were collected, fixed for 1 h in 2% paraformaldehyde at room temperature, permeabilized for 30 min in 0.1% sodium citrate, 0.1% Triton X-100 in PBS at room temperature, and subjected to TUNEL reaction using the In situ cell death detection kit, TMR-Red (Roche, Laval, Quebec, Canada), following the manufacturer’s instruction. Labeled cells were analyzed using Cytomics FC 500 flow cytometer (Beckman-Coulter, Mississauga, Ontario, Canada) using FL2 filter (575 nm).

3-(4,5-Dimethyl-2-thiazolyl) (MTT) viability assay
Cells were plated in 96-well plates at a density of 1 x 104 cells per well and incubated overnight at 37 C after which they reached 80% confluence. Recombinant TNF{alpha} was added to selected wells at the indicated concentrations in 100 µl culture medium, and plates were incubated for indicated times at 37 C. MTT reagent (Sigma) was added to the wells (10 µl of a 0.5% solution in PBS) 3.5 h before the end of the incubation period, and conversion of yellow tetrazolium salt to blue thiazol crystals by metabolically active cells was stopped by adding 100 µl of a 10% sodium dodecyl sulfate, 0.1% HCl solution to each well. Plates were incubated overnight at 37 C to allow complete solubilization of thiazol crystals, and intensity of blue emission in each well was measured using FluoStar multiwell plate reader (BMG Laboratories, Durham, NC). Percentage of proliferating cells was calculated as the ratio of optical densities of treated to control-treated cells.

Western blot analysis
Treated cells were disrupted in cold RIPA buffer containing protease inhibitors (Complete from Roche, Laval, Quebec, Canada) followed by three freeze-thaw cycles. Equal amounts of cell lysates were separated onto 10–15% polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). The membranes were probed with primary antibody overnight at 4 C and incubated with horseradish peroxidase-conjugated secondary antibody for 45 min. Detection was performed using SuperSignal West Femto substrate (Pierce, Arlington Heights, IL), as described by the manufacturer.

Targeting of XIAP by siRNA
Cells were seeded in six-well plates at a density of 2.5 x 105 cells per well and allowed to adhere overnight. The day of the experiment, XIAP (5'-ccaagugguaguccuguuucagcau-3' and 5'-augcugaaacaggacuaccacuugg-3') or control (5'-acucuaucugcacgcugacuu-3' and 5'-aagucagcgugcagauagagu-3') siRNAs were mixed with Trans-it TKO transfection reagent (Mirus, Madison, WI) following supplier’s instructions and added to the cells (100 nM working concentration). Plates were incubated for 24 h at 37 C, and medium was replaced with fresh medium containing TNF{alpha} or other agents as indicated in the figure legends. Plates were incubated for 24 h at 37 C before cells were collected.

Immunofluorescence-based detection of XIAP in clinical samples
Human endometrial carcinoma tissue section slide (Cybrdi, Frederick, MD), containing 17 grade I tumor specimens, 33 grade II tumor specimens, 5 grade III tumor specimens and 3 normal endometrial specimens, was used. The tissues, obtained from biopsies, were already formalin-fixed and paraffin-embedded. Slides were deparaffinized by heating at 60 C for 30 min followed by two washes in NeoClear solvent (VWR Canlab, Mississauga, Ontario, Canada), and progressively hydrated. After permeabilization in boiling citrate solution (0.1% sodium citrate, 0.1% Triton X-100 in water), nonspecific binding sites were blocked with goat serum, and tissues were probed with rabbit anti-XIAP primary antibody overnight at 4 C. Negative control staining was obtained by replacing primary antibody with 2% bovine serum. Tissues were probed with Alexa Fluor 488-conjugated antirabbit secondary antibody (Cell Signaling) for 1 h and counterstained with Hoechst nuclear dye. Tissues were covered with Prolong antifade mounting medium (Invitrogen) and observed under a fluorescence microscope.

Statistical analysis
Data were subjected to one-way ANOVA (PRISM software version 3.03; GraphPad, San Diego, CA). Differences between experimental groups were determined by the Tukey’s posttest. Statistical significance was accepted when P < 0.05. Where appropriate, data were also analyzed using two-way ANOVA (PRISM) to test for interactions between proteins or treatments, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether TNF{alpha} can induce apoptosis in endometrial carcinoma cells, we used Ishikawa and Hec-1A cell lines as study model. Both cell lines constitutively produce TNF-R1 transcript (Fig. 1AGo), as well as TNF-R1 protein at the plasma membrane (Fig. 1BGo) and do not produce detectable levels of TNF-R2 transcript, under basal conditions or after ligand stimulation (Fig. 1AGo). Using Hoechst nuclear staining as well as TUNEL assay, we found that TNF{alpha} could induce apoptosis in Ishikawa cells (Fig. 1Go, C–F). Apoptosis induction was optimal at 20 ng/ml TNF{alpha} (Fig. 1Go, C and E), as soon as 24 h after treatment (Fig. 1Go, D and F). However, we observed that a concentration of 4 ng/ml TNF{alpha} was sufficient to induce a general decrease of cellular viability in Ishikawa cells after 24 h of treatment (Fig. 1GGo). This suggests that apoptotic events do not contribute to the observed decrease of viability induced by a suboptimal dose of TNF{alpha}. Nonetheless, the decrease of viability of Ishikawa cells caused by the different doses of TNF{alpha} after the first 24 h of treatment was higher than the decrease that occurred during the next 24 h period (interaction, P < 0.05, two-way ANOVA), indicating that similar to apoptosis induction, viability reduction was optimal after 24 h of treatment with TNF{alpha} in Ishikawa cells. Therefore, based on the above-mentioned results, a 24-h treatment with 20 ng/ml TNF{alpha} were the conditions chosen for subsequent experiments. Importantly, we found that TNF{alpha} was not able to induce apoptosis in Hec-1A cells, even when the concentration of the cytokine was increased up to 100 ng/ml (Fig 1Go, C and E) or after a prolonged treatment of 72 h (Fig. 1Go, D and F). In addition, these cells showed no significant decrease of cellular viability (Fig. 1HGo), even after 72 h of exposure to the cytokine. These results indicate that, contrary to Ishikawa cells, Hec-1A cells are intrinsically resistant to TNF{alpha}-induced apoptosis. Noteworthy because Hoechst nuclear staining and TUNEL analysis generated comparable results in the above-mentioned experiments (Fig. 1Go, C–F), we chose to perform only Hoechst nuclear staining for subsequent apoptosis analyses.


Figure 1
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  		FIG. 1. TNF{alpha} dose-dependently induces apoptosis in Ishikawa cells. A, TNF-R1 and TNF-R2 mRNA expression was assessed in Ishikawa and Hec-1A cells using RT-PCR. cDNA from Jurkat cells was used as a positive control, and GAPDH was used as a loading control. Results are representative of three independent experiments. B, TNF-R1 expression on plasma membrane of Ishikawa and Hec-1A cells was determined using flow cytometry. Control labeling was performed using 2% bovine serum. Results are representative of three independent experiments. C–F, Ishikawa and Hec-1A cells were treated with increasing doses of recombinant TNF{alpha} for 24 h (C and E) or with 20 ng/ml TNF{alpha} for increasing time periods (D and F) and apoptosis index was determined in treated cells using Hoechst nuclear staining (C and D) or TUNEL assay (E and F). Results are mean ± SEM of three independent experiments. G and H, Ishikawa (G) and Hec-1A cells (H) were treated with increasing doses of TNF{alpha} for increasing time periods as indicated, and subjected to MTT viability assay. Results are mean ± SEM of three independent experiments. *, P < 0.05 compared with untreated (control) cells. {dagger}, P < 0.05 compared with untreated (control) cells after 24 h of treatment. §, P < 0.05 compared with untreated (control) cells after 48 h of treatment. {ddagger}, P < 0.05 compared with untreated (control) cells after 72 h of treatment.

 
To determine whether TNF{alpha} failed to induce apoptosis in Hec-1A cells because of altered caspase cascade, we have compared caspase signaling activated by the cytokine in sensitive Ishikawa cells and resistant Hec-1A cells. In Ishikawa cells, caspases-8, -9, and -3 were efficiently cleaved/activated in response to TNF{alpha}, leading to the cleavage of DNA repair protein PARP (Fig. 2AGo). Blockade of TNF-R1 using a neutralizing antibody completely prevented TNF{alpha}-induced cleavage of caspases-8, -9, and -3, and PARP (Fig. 2BGo) as well as the induction of apoptosis (Fig. 2CGo), indicating that TNF{alpha} properly activates caspase cascade in a TNF-R1-dependent manner in sensitive Ishikawa cells. In TNF{alpha}-resistant Hec-1A cells, cleavage/activation of caspase-8 also occurred in response to TNF{alpha} (Fig. 2DGo) and could be prevented using TNF-R1 blocking antibody (Fig. 2EGo). However, caspase-8 cleavage was delayed in these cells because it was not detectable before a 48-h exposure (Fig. 2DGo). In addition, TNF{alpha} could not activate caspase-9 and -3 and did not cause PARP cleavage/degradation in Hec-1A cells, even after a 72-h exposure to the cytokine (Fig. 2DGo), indicating that particular cellular mechanisms interfere with caspase cleavage/activation downstream of TNF-R1-induced caspase-8 activation in these cells.


Figure 2
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  		FIG. 2. TNF-R1-induced caspase signaling is altered in Hec-1A cells. A and D, Ishikawa (A) and Hec-1A cells (D) were treated with 20 ng/ml TNF{alpha} for increasing time periods and caspase-8, -9, -3 and PARP cleavage was monitored using Western blot. β-Actin was used as a loading control. B and C, Ishikawa cells were pretreated with neutralizing anti-TNF-R1 antibody (0, 1, 10 µg/ml) for 1 h before treatment with 20 ng/ml TNF{alpha} for 24 h and (B) caspase-8, -9, -3 and PARP cleavage was monitored using Western blot and (C) apoptosis was quantified using Hoechst nuclear staining. Results are mean ± SEM of three independent experiments. *, P < 0.05 compared with untreated (control) cells. E, Hec-1A cells were pretreated with neutralizing anti-TNF-R1 antibody (0, 1, and 10 µg/ml) for 1 h before treatment with 20 ng/ml TNF{alpha} for 72 h and caspase-8 cleavage was monitored using Western blot. All results are representative of three independent experiments. cl. casp, Cleaved caspase.

 
In a search for factors involved in such interference with caspase signaling in TNF{alpha}-resistant cells, we have investigated a role for Akt. Indeed, increased Akt activity can block caspase-8-mediated apoptosis (19), and Akt activation by TNF{alpha} has been shown to counteract TNF{alpha}-induced apoptosis (10). We had previously showed that Ishikawa cells are P-Akt-positive due to mutated phosphatase and tensin homolog (PTEN) status, whereas Hec-1A cells express a WT PTEN protein and are P-Akt-negative (20). Because P-Akt-positive Ishikawa cells were TNF{alpha}-sensitive (Fig. 1Go, C–F), and knowing that proapoptotic factors can decrease P-Akt levels in cancer cells (17), we hypothesized that exposure to TNF{alpha} down-regulates P-Akt levels in sensitive Ishikawa cells, and therefore prevents the prosurvival kinase from protecting the cells against TNF{alpha}-induced apoptosis. Our results showed that endogenous levels of phosphorylated/active Akt, as well as total Akt content, were reduced after treatment with TNF{alpha} in Ishikawa cells (Fig. 3Go, A and C). Statistical analysis revealed that Akt and P-Akt levels were decreased similarly by TNF{alpha} (no interaction, P > 0.05, two-way ANOVA), strongly suggesting that the decrease of phosphorylated/active form of Akt does not result from a change in phosphorylation/dephosphorylation of Akt, but rather results from the decrease of total Akt content. In P-Akt-negative Hec-1A cells, exposure to TNF{alpha} did not modulate total Akt content nor induce the phosphorylation of Akt (Fig. 3Go, B and D). Altogether, these results strongly suggest that Akt activity is insufficient in endometrial carcinoma cells in response to TNF{alpha} to confer resistance to TNF{alpha}-induced apoptosis, and that factors different from Akt are responsible for altered caspase signaling in resistant Hec-1A cells.


Figure 3
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  		FIG. 3. XIAP RNAi sensitizes endometrial carcinoma cells to TNF{alpha}. A–D, Ishikawa (A and C) and Hec-1A (B and D) cells were treated with 20 ng/ml TNF{alpha} for 24 h and P-Akt, Akt, and XIAP levels were determined using Western blot (A and B) and densitometric analyses of the blots showed in A and C were performed (C and D). Results are mean ± three independent experiments. *, Statistically different than control cells (P < 0.05). E–H, XIAP was silenced using RNAi in Ishikawa (E and G) and Hec-1A cells (F and H) before treatment with 20 ng/ml TNF{alpha} for 24 h. XIAP content and caspase-8, -9, -3. and PARP cleavage was monitored using Western blot (E and F) and apoptosis was quantified using Hoechst nuclear staining (G and H). Results are mean ± SEM of three independent experiments; *, P < 0.05; {dagger}, Normalized P-Akt levels were significantly different (P < 0.05) in treated compared with untreated (control) cells. §, Normalized XIAP levels were significantly different (P < 0.05) in treated compared with untreated (control) cells. {ddagger}, Normalized total Akt levels were significantly different (P < 0.05) in treated compared with untreated (control) cells. Ctl, Control; cl. casp, cleaved caspase.

 
We have recently observed that XIAP can block caspase signaling in endometrial carcinoma cells treated with TGF-β (16), and considering that XIAP can confer resistance to TNF{alpha}-induced apoptosis in non-cancer cells (18), we have examined the role of XIAP and its interaction with caspase signaling in endometrial cancer cells in response to TNF{alpha}. We found that protein levels of XIAP were decreased in TNF{alpha}-sensitive Ishikawa cells (Fig. 3Go, A and C) but increased in TNF{alpha}-resistant Hec-1A cells (Fig. 3Go, B and D). In light of these results, we hypothesized that such an increase of XIAP protein levels in Hec-1A cells could confer resistance to TNF{alpha}-induced apoptosis through its caspase-inhibitory activity. Indeed, we found that knockdown of XIAP by siRNA in Hec-1A cells allowed TNF{alpha} to trigger caspases and PARP cleavage (Fig. 3FGo), and to induce apoptosis (Fig. 3HGo), as early as 24 h from the start of the treatment. In addition, silencing of XIAP in Ishikawa cells using RNA interference increased the ability of TNF{alpha} to induce caspases-8, -9, and -3, and PARP cleavage (Fig. 3EGo), and increased the extent of apoptosis induced by the cytokine (Fig. 3GGo). Moreover, statistical analysis suggested the presence of a synergistic effect of XIAP knockdown and TNF{alpha} on apoptosis induction in both cell lines (highly significant interaction, P < 0.001, two-way ANOVA). Altogether, these results indicate that XIAP levels regulate the sensitivity to TNF{alpha}-induced apoptosis in endometrial carcinoma cells.

Because increased XIAP levels confer resistance to TNF{alpha}-induced apoptosis in endometrial carcinoma cells (Fig. 3Go), factors involved in XIAP up-regulation by TNF{alpha} could putatively be targeted to increase cellular sensitivity to the cytokine. To identify these factors, we first examined whether XIAP regulation by TNF{alpha} occurred at the transcriptional level. In Ishikawa cells, exposure to TNF{alpha} decreased XIAP transcript levels in a time-dependent manner (Fig. 4Go, A and B); this decrease was detectable after 6 h of exposure to the cytokine, consistent with a decrease in XIAP protein levels occurring after 24 h of treatment (Fig. 3Go, A and C). Conversely, XIAP transcripts were increased in Hec-1A cells in response to TNF{alpha}, in a time-dependent manner (Fig. 4Go, A and B). This increase was also detectable after 6 h of exposure to the cytokine (Fig. 4Go, A and B), consistent with an increase of XIAP protein levels occurring after 24 h of treatment (Fig. 3Go, B and D). These results indicate that TNF{alpha} modulates XIAP content by acting at the gene expression level in endometrial carcinoma cells. Because TNF{alpha} can activate ERK, PI3-K and PKC signaling pathways (9, 10), we hypothesized that at least one of these pathways could be involved in the up-regulation of XIAP expression by TNF{alpha} in Hec-1A cells. To examine this question, we selectively blocked these pathways using pharmacological inhibitors: constitutive activity of ERK pathway in Hec-1A cells, as evidenced by basal phosphorylation of P44/42, could be inhibited by treating the cells for 1 h with 10 µM MEK1 inhibitor PD98059 (Fig. 4CGo), whereas constitutive activity and phosphorylation of multiple PKC enzymes could be reduced by treating the cells for 1 h with PKC inhibitors chelerythrine chloride (0.2 µM) (Fig. 4CGo) and calphostin C (2 µM) (Fig. 5BGo). Absence of detectable Akt phosphorylation is a strong indicator of an inactive PI 3-K pathway in endometrial carcinoma cells (20), and Akt phosphorylation was not detectable in Hec-1A cells under basal conditions and also after a 1-h treatment with 10 µM PI 3-K inhibitor LY294002 (Fig. 4CGo). On the opposite, Akt is constitutively phosphorylated in Ishikwawa cells, due to mutated PTEN protein (20), and Akt phosphorylation could be inhibited by treating these cells for 1 h with 10 µM PI 3-K inhibitor LY294002 (Fig. 4CGo). Similar to Hec-1A cells, constitutive ERK and PKC activities in Ishikawa cells could be inhibited using 10 µM PD98059 and 0.2 µM chelerythrine chloride, respectively (Fig. 4CGo). Noteworthy, we observed that similar to endogenous XIAP mRNA levels (data not shown), endogenous XIAP proteins levels remained unchanged in Hec-1A cells after inhibition of ERK, PI3-K or PKC activity, whereas they were reduced in Ishikawa cells after selective inhibition of PI3-K activity (Fig. 4Go, C and D). These results indicate that PI3-K activity is involved in the constitutive expression of XIAP in WT PTEN/P-Akt-positive Ishikawa cells and suggest that, in Hec-1A cells, constitutive expression of XIAP is not solely dependent on the activity of ERK, PKC, or PI3-K activity. Other pathways may be involved, and it is possible that two or more pathways converge to the promoter of XIAP for its regulation in resting Hec-1A cells.


Figure 4
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  		FIG. 4. TNF{alpha} increases XIAP mRNA levels through PKC activity in resistant Hec-1A cells. A, Ishikawa and Hec-1A cells were treated with 20 ng/ml TNF{alpha} for the indicated time periods and XIAP transcript levels were determined using RT-PCR. GAPDH was used as a loading control. B, Densitometric analyses of the results presented in A. C, Ishikawa and Hec-1A cells were treated for 1 h with 10 µM PI 3-K inhibitor LY294002, 10 µM MEK inhibitor PD98059 or 0.2 µM PKC inhibitor (inhib.) chelerythrine chloride, and efficient inhibition of Akt phosphorylation, ERK phosphorylation and PKC phosphorylation, respectively, as well as XIAP content, was monitored using Western blot. β-Actin was used as a loading control. Positive control: total extract from Ishikawa cells. D, Densitometric analysis of the results presented in C. E, Hec-1A cells were pretreated for 1 h with 10 µM MEK inhibitor PD98059, 0.2 µM PKC inhibitor chelerythrine chloride, or 10 µM PI 3-K inhibitor LY294002 before treatment with 20 ng/ml TNF{alpha} for 24 h and XIAP transcript levels were determined using RT-PCR. GAPDH was used as a loading control. c.c., Calphostin C. F, Densitometric analysis of the results presented in D.

 

Figure 5
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  		FIG. 5. Inhibition of PKC activity sensitizes endometrial carcinoma cells to TNF{alpha}-induced apoptosis. A and B, Hec-1A cells were pretreated for 1 h with 0.2 µM chelerythrine chloride (A) or 2 µM casphostin C (B) before treatment for 24 h with 20 ng/ml TNF{alpha} and XIAP, P-PKC, cleaved caspase-3 and cleaved PARP fragments were monitored using Western blot, whereas apoptosis was quantified using Hoechst nuclear staining. C, Ishikawa cells were pretreated for 1 h with 0.2 µM chelerythrine chloride before treatment for 24 h with 20 ng/ml TNF{alpha} and XIAP, P-PKC, cleaved caspase-3, and cleaved PARP fragments were monitored using Western blot, whereas apoptosis was quantified using Hoechst nuclear staining. Results are representative or mean of three independent experiments. cl. casp, Cleaved caspase.

 
Then, to determine whether TNF{alpha} induces XIAP expression in Hec-1A cells by acting on these pathways, cells were pretreated with pharmacological inhibitors as described above, before they were treated with TNF{alpha}. We found that the up-regulation of XIAP transcript levels by TNF{alpha} was not prevented when MEK/ERK or PI3-K pathways were inhibited (Fig. 4Go, E and F); however, it was specifically blocked by two different large-spectrum PKC inhibitors, chelerythrine chloride, (which inhibits PKC {alpha}, β, {delta}, and {gamma}), and calphostin C (which inhibits PKC {eta} and {theta} in addition to PKC {alpha}, β, {delta}, {epsilon}, and {gamma}) (Fig. 4Go, E and F). These results indicate a role for PKCs in TNF{alpha}-induced XIAP mRNA up-regulation in Hec-1A cells. Pretreatment with chelerythrine chloride (Fig. 5AGo) or calphostin C (Fig. 5BGo) also blocked TNF{alpha}-induced up-regulation of XIAP protein in Hec-1A cells; in fact, PKC inhibition allowed TNF{alpha} to down-regulate XIAP protein, to induce the cleavage of caspase-3 and PARP, and to induce apoptosis in Hec-1A cells (Fig. 5Go, A and B). Similarly, pretreatment of Ishikawa cells with chelerythrine chloride increased the impact of TNF{alpha} on XIAP down-regulation, on the generation of cleaved caspase-3 and PARP fragments, and on the induction of apoptotic cells (Fig. 5CGo). Statistical analysis suggested the presence of a synergistic effect of PKC inhibition and TNF{alpha} on apoptosis induction in both cell lines (highly significant interaction, P < 0.001, two-way ANOVA). Altogether, these results indicate that PKC inhibition allows TNF{alpha} to down-regulate XIAP protein and potentiates the proapoptotic effect of TNF{alpha} in endometrial carcinoma cells.

We have shown that TNF{alpha} can efficiently induce apoptosis in endometrial carcinoma cells (Fig. 1Go) and that direct or indirect reduction of XIAP levels increases cellular sensitivity to the cytokine (Figs. 3–5GoGoGo), at least in vitro. Before initiating the study of a similar role for XIAP in vivo, it is necessary to first ensure that XIAP is indeed expressed by tumor cells from human endometrial tumor tissue samples. To this aim, we have conducted immunofluorescence analysis on a large number of human endometrial carcinoma tissue samples representing various stages of the disease (Fig. 6Go). Our results showed the presence of XIAP immunoreactivity in epithelial cells from stage I (Fig. 6AGo) and stage II (Fig. 6BGo) endometrial carcinoma tissues, as well as from normal endometrium tissue (Fig. 6DGo). XIAP immunoreactivity was almost absent in stage III endometrial carcinoma tissues (Fig. 6CGo). Altogether, these results indicate that XIAP is indeed expressed by endometrial carcinoma cells in vivo, except in late-stage tumors.


Figure 6
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  		FIG. 6. XIAP is present in endometrial carcinoma cells in vivo. A–D, XIAP immunoreactivity was determined (green) in human endometrial carcinoma tissues grade I (17 specimens) (A), grade II (33 specimens) (B) and grade III (5 specimens) (C), and normal endometrial tissue (D). E and F, Negative staining (2% bovine serum). Nuclei were counterstained with Hoechst dye (blue). Green staining (F) is due to autofluorescence of erythrocytes that are present in the lumen of blood vessels Magnification, x400.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several anti-cancer therapies based on the antitumor effects of TNF{alpha} are now in clinical trial (reviewed in Ref. 11). Endometrial tumors often show resistance to current chemotherapeutic drugs (21), which induce apoptosis via the intrinsic pathway, and TNF{alpha} could constitute an interesting alternative therapeutic approach because it induces apoptosis via extrinsic death receptor-activated pathway. We have evaluated the proapoptotic effect of TNF{alpha} in endometrial carcinoma cells, using two well-established model cell lines: Ishikawa and Hec-1A cells (22, 23, 24). We show for the first time that TNF{alpha} can indeed induce apoptosis in endometrial carcinoma cells, although only one of the two TNF-R1-expressing cell lines studied was intrinsically sensitive to TNF{alpha}-induced apoptosis (Ishikawa cells). This observation is consistent with the fact that sensitive Ishikawa cells were derived from a well-differentiated (grade I) tumor, whereas resistant Hec-1A cells were derived from a poorly differentiated tumor (grade 3). We have dissected molecular mechanisms underlying such difference in sensitivity to TNF{alpha}-induced apoptosis between the two model cell lines, which are genetically unrelated, and among other factors, we found that in Hec-1A cells, resistance to TNF{alpha}-induced apoptosis was associated with delayed TNF-R1-dependent caspase-8 activation. In ovarian cancer cells, TNF{alpha} has been shown to increase the levels of Fas-asscoiated death domain-like IL-1-converting enzyme-like inhibitory protein [FLIP(s)], an antiapoptotic protein that acts in a dominant-negative manner to prevent caspase-8 recruitment to death receptors and its subsequent activation (25, 26); increased FLIP(s) expression has been proposed to contribute to the resistance of ovarian epithelial cancer cells to TNF{alpha}-induced apoptosis (27). Therefore, TNF{alpha} could also increase FLIP(s) expression in endometrial carcinoma cells; this could explain the observed delay of caspase-8 activation by the cytokine, and thereby contribute to the resistance of Hec-1A cells to TNF{alpha}-induced apoptosis.

We have found that resistance to TNF{alpha}-induced apoptosis in Hec-1A cells not only involves delayed activation of caspase-8, but also a disruption of caspase signaling downstream of caspase-8. We have examined a putative role for Akt in altered caspase signaling because increased Akt activity can block caspase-8-mediated apoptosis (19), and knowing that Akt plays a key role in endometrial cancer cell survival in response to proapoptotic agents (28). The two endometrial carcinoma cell lines Ishikawa and Hec-1A differ with respect to constitutive activity of PI3-K pathway: we had previously showed that Ishikawa cells are P-Akt-positive due to mutated PTEN status, whereas Hec-1A cells harbor a WT PTEN protein and are P-Akt-negative (20). Considering the general prosurvival role for Akt, one could expect P-Akt-positive Ishikawa cells to be more resistant than P-Akt-negative Hec-1A cells to proapoptotic factors such as TNF{alpha}. However, proapoptotic factors often directly modify cellular P-Akt content, thereby modifying cellular sensitivity to apoptosis. TNF{alpha} has already been shown to modify P-Akt levels in a cell-specific manner: for example, the cytokine increases P-Akt levels in colon and cervical cancer cells (29) but decrease the levels of P-Akt in adipocytes (30). We show here that TNF{alpha} can also modify P-Akt content in endometrial carcinoma cells; both P-Akt and total Akt levels were similarly decreased by the cytokine in P-Akt-positive Ishikawa cells. Considering that TNF{alpha} also activates caspase signaling in these cells, and because we have shown that Akt is a substrate for caspase-3 (31, 32), caspase-3 activity may be responsible for the decrease of Akt in response to TNF{alpha} in Ishikawa cells. Overall, because Akt is targeted by TNF{alpha} in P-Akt-positive Ishikawa cells, phosphorylated/active Akt levels are probably insufficient to block caspase signaling in response to TNF{alpha} in these cells, similar to Hec-1A cells where P-Akt is undetectable, even in response to TNF{alpha}.

We have recently shown evidence of a key antiapoptotic role for XIAP in endometrial carcinoma cells. In particular, we have observed that, in these cells, XIAP can block TGF-β-induced caspase signaling (16), as well as apoptosis induced by chemotherapeutic drugs (17). Because XIAP has been reported to confer resistance to TNF{alpha}-induced apoptosis in non-cancer cells (18), we have investigated a putative role for this antiapoptotic protein in blocking TNF{alpha}-induced caspase signaling in endometrial carcinoma cells. XIAP mRNA and protein levels are increased in these cells in response to TNF{alpha}, probably in a NF-{kappa}B-dependent manner as was shown in other cell types (18, 33). There is evidence that such an increase of XIAP induced by TNF{alpha} and involving NF-{kappa}B could protect cells against TNF{alpha}-induced apoptosis (27). In the present study, we provide direct demonstration that XIAP up-regulation by TNF{alpha} blocks caspase signaling and protects endometrial carcinoma cells against TNF{alpha}-induced apoptosis. XIAP has been shown to regulate cellular sensitivity to multiple apoptosis-inducing agents in cancer cells (16, 34), but this is the first demonstration, to our knowledge, of a direct role for XIAP in cancer cell resistance to apoptosis induced by TNF{alpha}.

XIAP is a ubiquitous protein which plays a key role in controlling the balance between cell survival/apoptosis because of its ability to bind and inhibit caspases (35). In the normal cycling endometrium, where tightly regulated apoptotic events take place, XIAP has been shown to be present at all times [at least in the rat model (36)]. It is therefore not surprising that XIAP immunoreactivity could also be detected in normal human endometrium. XIAP and its inhibitors, such as Smac/Diablo (37) and XAF-1 (38), probably play a central role in determining the fate of normal and cancerous human endometrial cells. In this regard, we found XIAP immunoreactivity in most tumor cells within human tumor tissue samples, and therefore XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNF{alpha}-induced apoptosis in endometrial tumor cells. Surprisingly, XIAP was almost absent in tumor cells from stage III tumors; this suggests that late-stage endometrial carcinoma cells have developed other mechanisms of resistance to caspase-mediated apoptosis. Altered activation of caspase signaling in response to proapoptotic stimuli, for example, could allow cancer cells to overcome the need for XIAP. In this regard, we have shown recently that TGF-β cannot induce caspase signaling in endometrial carcinoma cells, which are intrinsically resistant to TGF-β-induced apoptosis (16). The underlying mechanism is still unknown, but it is possible that, similar to other cancer cell types, endometrial carcinoma cells express a mutated TGF-β receptor I (39) or receptor II (40) protein that in many cases is truncated and not present at the cell surface (40). Such inactivating mutations of caspase-activating receptors, as well as down-regulation of death receptors expression (41), represent a very efficient means for cancer cells to prevent caspase-dependent apoptosis and therefore, to bypass a need for XIAP. Others have shown that the expression of FLIP, which competes with caspase-8 for recruitment to death receptor and prevents subsequent caspase-8 activation, is frequent in endometrial tumor cells and correlates with staging (21). FLIP expression has been shown to confer protection against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) (21, 42) and Fas (42) in endometrial carcinoma cell lines and has been postulated to protect other cancer cells against TNF{alpha}-induced apoptosis (27). TNF{alpha} can induce the expression of FLIP in cancer cells (27) and therefore, constitutive FLIP expression is not a requirement for these cells to evade TNF{alpha}-induced apoptosis. Similarly, TNF{alpha} has been reported to increase the expression c-IAP1 and cIAP-2, which blocks caspase signaling and confers resistance to TNF{alpha}-induced apoptosis (43). Inducible up-regulation of caspase-inhibitory factors could therefore represent another mechanism used by cancer cells to overcome the need for XIAP.

Importantly, we found that inhibition of PKC activity sensitizes endometrial carcinoma cells to TNF{alpha}-induced apoptosis. PKC inhibition has also been shown to sensitize other types of cancer cells to apoptosis induced by TNF{alpha} (44), and also by TRAIL (45). Noteworthy, in uterine (cervical) cancer cells HeLa, PKC inhibition induces XIAP protein degradation which sensitizes the cells to TRAIL-induced apoptosis (45). In endometrial carcinoma cells, PKC inhibition itself has no impact on XIAP gene expression or protein content, and this suggests that regulation of endogenous XIAP protein content by PKC activity is cell type specific. Rather, PKC inhibition indirectly increases TNF{alpha}-induced XIAP down-regulation, which potentiates TNF{alpha}-induced apoptosis. Overall, this strengthens our conclusion that XIAP protein levels regulate the sensitivity of endometrial carcinoma cells to TNF{alpha}-induced apoptosis.

The presence of XIAP in tumors cells from clinical samples of endometrial carcinoma, and our observation that XIAP regulates the sensitivity of endometrial carcinoma cells to TNF{alpha}-induced apoptosis in vitro, urges the need for additional in vivo studies aimed at determining the regulation of XIAP throughout endometrial carcinoma progression and its role in TNF{alpha}-induced apoptosis in vivo. Several in vivo studies have shown that TNF{alpha} was more efficient when used in combination therapy (46, 47). Thus, administration of recombinant TNF{alpha} in combination with PKC inhibitors or other agents targeting XIAP could therefore prove highly efficient for the treatment of endometrial cancer.

In conclusion, we showed for the first time that TNF{alpha} can efficiently induce apoptosis in endometrial carcinoma cells. We have also identified a key role for XIAP in regulating cellular sensitivity to TNF{alpha}-induced apoptosis, and highlighted the potent sensitizing effect of PKC inhibition. Local administration of recombinant TNF{alpha}, in combination with PKC inhibitors or other agents targeting XIAP, may therefore represent an alternative therapeutic approach for the treatment of endometrial cancers.


   Acknowledgments
 
The authors want to thank Mrs. Valérie Leblanc and Amélie Journault for technical assistance.


   Footnotes
 
This work has been supported by a grant from the Canadian Institutes for Health Research (MOP-66987). C.V.T. is a holder of a postdoctoral fellowship from the Cancer Research Society (Montreal). E.A. holds a Canada Research Chair in Molecular Gyneco-Oncology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2008

Abbreviations: AS, Antisense; FLIP, Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEK, MAPK kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl); NF-{kappa}B, nuclear factor-{kappa}B; PARP, poly(ADP-ribose) polymerase; PI3-K, phosphatidylinositide 3-kinase; PKC, protein kinase C; P-PKC, phospno-PKC; PTEN, phosphatase and tensin homolog; S, sense; TNF-R1 and TNF-R2, two TNF receptors; TRAIL, TNF-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphosphate nick-end labeling; XIAP, X-linked inhibitor of apoptosis protein.

Received February 28, 2008.

Accepted for publication April 30, 2008.


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