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Carcinogenesis Advance Access originally published online on December 19, 2005
Carcinogenesis 2006 27(5):936-944; doi:10.1093/carcin/bgi316
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Overexpression of thymosin ß-4 renders SW480 colon carcinoma cells more resistant to apoptosis triggered by FasL and two topoisomerase II inhibitors via downregulating Fas and upregulating Survivin expression, respectively

Hung-Liang Hsiao, Wei-Shu Wang 1, Po-Min Chen 1 and Yeu Su 2, *

Institute of Pharmacology, College of Medicine, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan, R.O.China, 1 Division of Medical Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.China and 2 Institute of Biopharmaceutical Science, Collage of Life Science, National Yang-Ming University, Taipei, Taiwan, R.O.China

* To whom correspondence should be addressed. Email: yeusu{at}ym.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present work was conducted to further examine the effects of thymosin ß-4 (Tß4) upregulation on the apoptosis of SW480 colon cancer cells induced by T cells and various chemotherapeutic agents because reduced susceptibility to the cytotoxicity of an anti-Fas IgM (CH-11) in Tß4-overexpressing cells has previously been reported by us. As expected, Tß4 overexpressers were also more resistant to the killing effect of FasL-bearing Jurkat T cells. On the other hand, pretreating these cells with an MMP inhibitor restored not only their Fas levels but also their sensitivity to CH-11, suggesting a pivotal role of MMP in downregulating Fas in Tß4 overexpressers. Interestingly, while the susceptibilities of Tß4 overexpressers to 5-FU and irinotecan remained unchanged, they were more resistant to doxorubicin and etoposide which triggered apoptosis via a mitochondrial pathway. Concordantly, activation of both caspases 9 and 3 in Tß4 overexpressers by the two aforementioned topoisomerase II inhibitors was dramatically abrogated which could be accounted mainly by an increased expression of Survivin, a critical anti-apoptotic factor. Finally, poor survival was found in stage III colon cancer patients whose tumors were stained positively by the anti-Survivin antibody. Thus, advantages such as immune evasion and resistance to anticancer drug-induced apoptosis acquired by colon cancer cells through Tß4 overexpression might facilitate their survival during metastasis and chemotherapy.

Abbreviations: CRC, colorectal carcinoma; CTLs, cytotoxic T lymphocytes; cyt c, cytochrome c; DR, death receptor; FasL, Fas ligand; IHC, Immunohistochemical; LAK, lymphokine-activated killer; MMP, matrix metalloprotease; MTT assay, 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-2H tetrazolium bromide assay; NK, natural killer; NSAIDs, non-steroidal anti-inflammatory drugs; Tß4, thymosin ß-4; TRAIL, TNF-related apoptosis-inducing ligand


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Interactions between the immune system and malignant cells play an important role in tumorigenesis. Failure of the immune system to detect and eliminate transformed cells may lead to cancer development (1). In this regard, mechanisms such as immunological ignorance, impaired antigen presentation, expression of immunosuppressive molecules and tolerance induction have been reported to facilitate evasion of various tumors from immune-mediated rejection (2). Escape from the immunosurveillance of cancers could be augmented by their resistance to the cytotoxic T lymphocytes (CTLs), natural killer (NK) and lymphokine-activated killer (LAK) cells. Besides the granule exocytosis pathway, immune cells kill tumors by activating their death receptors via specific ligands. For example, CTLs, NK and LAK cells utilize Fas ligand (FasL) to trigger apoptosis by interacting with Fas on the target cells (3–5). NK cells, in addition, can use TRAIL (TNF-related apoptosis-inducing ligand) to initiate apoptosis of the target cells through death receptors DR4 and DR5 (6). In addition to signaling through death receptors (extrinsic pathway), certain cellular stresses stimulate the release of cytochrome c (cyt c), Smac/Diablo or Omi/HtrA2 from mitochondria, which activate caspase 9, leading to the cleavage and activation of effector caspases (caspases 3, 6 and 7) to execute the death commands (7). Interestingly, the mitochondrial (intrinsic) death pathway can also be initiated by the truncated Bid generated from caspase 8 cleavage (8), indicating a cross-talk between the extrinsic and the intrinsic pathways.

Chemotherapy is an essential part of colorectal carcinoma (CRC) treatment, which relies mainly on inducing death (apoptosis and necrosis) of tumor cells via different mechanisms. In this regard, both the extrinsic and the intrinsic pathways have been shown to be involved in the apoptosis of colon cancer cells triggered by various drugs used in CRC therapy. For instance, upregulated Fas expression and a consequential sensitization to the lytic effects of CTLs induced by 5-FU, cisplatin and irinotecan (CPT-11) were demonstrated in SW480 cells (9,10). Increased FasL and/or Fas expression seemed to be critical for the apoptosis of SW480 and HCT15 cells triggered by doxorubicin and oxaliplatin, respectively (11) In addition, the threshold required for TRAIL-induced caspase 8 activation as well as apoptosis in both HT29 and SW480 cells was lowered by doxorubicin and cisplatin (12). A p53-dependent upregulation of DR5 by etoposide and irinotecan appeared to be essential for sensitizing the HCT116 colon cancer cells to TRAIL (13). In contrast, a Fas-independent activation of caspase 8 during the apoptosis of HT29-D4 cells induced by anti-microtubule agents was reported (14). Apoptosis of HCT116 cells triggered by oxaliplatin seemed to be associated with the release of cyt c (15). Furthermore, the release of SMAC/Diablo from mitochondria was demonstrated to be critical for non-steroidal anti-inflammatory drugs (NSAIDs)-induced apoptosis of HCT116 cells (16). Despite some success in metastatic CRC therapy achieved using a new multi-drug regimen (17), the resistance to individual agents as well as their underlying mechanisms need to be resolved before higher response rate to chemotherapy could be accomplished.

We have previously demonstrated a downregulated Fas expression and a reduced susceptibility to the anti-Fas agonistic antibody in SW480 colon carcinoma cells overexpressing thymosin ß-4 (Tß4) (18). Besides colon cancer, upregulated expression of this major G-actin sequestering peptide (19) has been correlated positively with the metastatic capacity of melanoma (20,21), fibrosarcoma (22) and oral squamous cell carcinoma (23). Except inducing changes in the cytoskeletal structure (24,25), the underlying mechanisms for facilitated metastasis and survival by Tß4 in these tumors are largely unknown. Since Fas is critical for transducing the death signals initiated by FasL as well as certain chemotherapeutic agents, both the major hurdles for metastasis and survival, in colon cancer cells, we therefore examined the apoptotic responses of the Tß4-overexpressing SW480 cells to FasL-bearing T cells and several anticancer drugs. We show that Tß4-overexpressers are more resistant to the cytotoxicity of Jurkat T cells and downregulation of Fas in these cells is primarily a result of matrix metalloproteinase (MMP)-7 cleavage. Additionally, these cells are killed less effectively by doxorubicin and etoposide, both induce apoptosis via a mitochondrial pathway. Finally, upregulation of Survivin not only is responsible for the drug resistance of the Tß4-overexpressers but is associated with a poor prognosis in Stage III CRC patients.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
Doxorubicin, etoposide and 5-FU were obtained from Pharmacia and Upjohn (Sweden). Irinotecan was purchased from Aventis (France). Geneticin (G418) was obtained from Calbiochem (San Diego, CA). L-15, RPMI culture medium and fetal calf serum (FCS) were purchased from Life Technologies (Rockville, MD). 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), caspase 9 inhibitor Z-LEHD-FMK and MMP inhibitor Galardin (GM6001) were obtained from Sigma (St Louis, MO). Caspases 9 and 3 colorimetric assay kits, FITC-conjugated goat anti-rabbit IgG and polyclonal anti-human Survivin antibodies were obtained from R&D Systems (Minneapolis, MN). Polyclonal antibodies against Fas, DR4 and ß-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-human caspase 9, Fas (agonistic, CH-11; neutralizing, ZB4) were purchased from Upstate (Lake Placid, NY). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Cell culture
Human CRC SW480 cells were maintained in L-15 medium supplemented with 10% FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C without CO2. Stable clones (480BK, 480S1 and 480S2) derived from the SW480 cells (26) were cultured in a similar condition except 0.8 mg/ml G418 was added into the medium. Jurkat T cells were maintained in RPMI medium supplemented with FCS, penicillin and streptomycin at 37°C with 5% CO2.

Cell viability assay
For analyzing cell viability after various treatments, 1 x 104 cells were seeded in 96-well plates containing 0.2 ml of medium. After 24 h the medium was replaced by the one containing either 1 x 104 fixed Jurkat cells [fixed with 2% paraformaldhyde in phosphate-buffered saline (PBS) at 4°C for 1 h], doxorubicin (0.125, 0.25, 0.5 and 1 µg/ml), etoposide (1.25, 2.5, 5 and 10 µg/ml), 5-FU (1.25, 2.5, 5 and 10 µg/ml) or irinotecan (5, 10, 20 and 40 µg/ml). After 48 h incubation, MTT assays were performed as described previously (27). To assess the effect of MMP inhibitor on FasL-induced apoptosis, cells were incubated with 50 µM GM6001 for 12 h before the addition of 40 ng/ml CH-11, the agonistic anti-Fas antibody. To evaluate the effects of neutralizing anti-Fas antibody on death triggered by drugs or FasL, cells were treated with 0.5 µg/ml of ZB4 for 1 h before the addition of doxorubicin (0.5 µg/ml), etoposide (5 µg/ml) or CH-11. For examining the protection of caspase 9 inhibitor against drug-induced apoptosis, cells were first incubated with 10 µM Z-LEHD-FMK and 12 h later doxorubicin or etoposide was added. MTT assays were conducted 48 h after the addition of aforementioned cytotoxic agents.

RT–PCR analysis
Preparation of total RNAs from both 480BK and 480S1 cells after they were incubated without or with doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) for 12, 24 and 48 h was performed as described previously (26). RT–PCR analysis was then conducted using the following primer sets: FasL (forward, 5'-CCACCACTGCCTCCACTACC-3'; reverse, 5'-CTGGGGATACTTAGAGTTCC-3'), Fas (forward, 5'-AGGGACTGCACAGTCAATGG-3'; reverse, 5'-CATGACTCCAGCAATAGTGG-3'), TRAIL (forward, 5'-GATCAAGACCATAGTGACCAA-3'; reverse, 5'-TGGCATGATCTCACCACAC-3'), DR4 (forward, 5'-GGTGCCCCCCCAAATTAAG-3'; reverse, 5'-TGAGGCTCACATGCCAAAGG-3'), DR5 (forward, 5'-CACGAGTGACACACTATACTATGGC-3'; reverse, 5'-CGACATGAAATAATTGGGGG-3'), Bcl-2 (forward, 5'-TGTCACAGAGGGGCTACGAG-3'; reverse, 5'-GGGCGATGTTGTCCACCAGG-3'), XIAP (forward, 5'-ATGTAGTGAAAGTTATGGTA-3'; reverse, 5'-CAACTGAGACTCAAAGGT-3'), Bcl-XL (forward, 5'-CATGGCAGCAGTAAAGCAAGC-3'; reverse, 5'-CTGCGATCCGACTCACCAATAC-3'), Survivin (forward, 5'-ATGGCACGGCGCACTTT-3'; reverse, 5'-TCCACTGCCCCACTGAGAA-3') and GAPDH (forward, 5'-GACCACAGTCCATGCCATCAC-3'; reverse, 5'-TCCACCACCCTGTTGCTGTAG-3'). All the reactions were performed based on the following program: 94°C for 10 min, 25–30 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 45 s and a final extension at 72°C for 7 min. PCR products were electrophoresed on a 2.5% agarose gel, followed by ethidium bromide staining and photographed under UV light.

Western blotting
Cell lysates were prepared as described previously (26) and 50 µg of proteins were separated on a 10% SDS–polyacrylamide gel and processed for immunoblotting, respectively, with anti-Fas, anti-caspase 9, anti-Survivin and ß-tubulin antibodies, and enhanced chemiluminescence reagent (ECL, NEN Life Science, Boston, MA) was used for signal detection.

Flow cytometric analysis
The cells were first fixed in 4% paraformaldehyde (in PBS) for 20 min at room temperature and then permeabilized in 0.1% Triton X-100 in Tris-buffered saline (TBS, pH 7.4) containing 1 mg/ml bovine serum albumin (BSA) for 5 min. Subsequently, these processed cells were reacted with 2 µg/ml of anti-Fas or anti-DR4 polyclonal antibodies for 20 min on ice, washed and then incubated with a 1:2000 dilution FITC-conjugated goat anti-rabbit IgG for 20 min. The flow cytometry was carried out with FACS Calibur (Becton Dickinson and Company, Oxford, CA) for relative protein content based on green fluorescence levels.

Caspase activation assays
Cells (2 x 106) after being treated with doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) for 24 h were harvested by scraping in a lysis buffer (caspase colorimetric assay kits, R&D Systems). Caspase 9 activity in 50 µg lysates was determined using LEHD-pNA as a substrate. For analyzing caspase 3 activity, 50 µg lysates prepared from cells 48 h after drug treatment were used in a reaction containing DEVD-pNA as a substrate. Reactions were carried out at 37°C for 2 h and enzyme activity was analyzed by measuring the 405 nm absorbance of each sample using a microplate reader (Model 550, Bio-Rad).

Plasmid construction and transient transfection
To construct a plasmid (pCMV-SurvivinAS) expressing the Survivin antisense RNA, the 274 bp Survivin DNA fragment obtained by RT–PCR using the forward primer 5'-gcgaattcATGGCACGGCGCACTTT-3' and the reverse primer 5'-gcggatccTCCACTGCCCCACTGAGAA-3' (sequence with underline represent restriction enzyme site) as above described was subcloned into the EcoRI and BamHI sites of the pBKCMV vector. Cells (3 x 105) were seeded in 6-well plates for 24 h before lipofectamine-mediated transient transfection was carried out with 4 µg of either the empty vector or the pCMV-SurvivinAS. After 24 h, transfected cells were trypsinized and seeded at a density of 1 x 104 cells/well in 96-well plates and doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) was added 24 h later. After another 48 h incubation, MTT assays were performed as above described. For western blot analysis, cell lysates were prepared 48 h post-transfection.

Immunohistochemical staining of Survivin and survival curve plotting
Between 2002 and 2003, a total of 41 patients with histologically confirmed Stage III CRC treated at Taipei Veterans General Hospital were enrolled. They all received curative tumor resection followed by 5-FU-based adjuvant chemotherapy. Immunohistochemical (IHC) staining of Survivin of the tissue samples was performed as described previously (28). Briefly, formalin-fixed, paraffin embedded tissue samples from these patients were sectioned, placed on slides and deparaffinized. A polyclonal anti-human Survivin antibody and a biotin-conjugated anti-rabbit IgG antibody were used as primary and secondary antibodies, respectively, followed by a sensitive peroxidase-conjugated streptavidin system (Dako, CA). Patients were then divided into two groups according to Survivin staining (positive or negative) and the survival curves were plotted using the Kaplan–Meier product limit method and data were analyzed by log-rank test (29).

Statistics
Each data point in the figures represents the mean ± SD for three individual determinations. Statistically significant differences (P < 0.05) between Tß4 transfected, vector-transfected and the parental SW480 cells were determined by one-way ANOVA and the Dunnett t-tests with repeated measures.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Tß4 overexpressers are more resistant to the cytolytic effects of Jurkat cells
Although Tß4-overexpressing SW480 cells have been shown to be less susceptible to the toxic effects of an anti-Fas IgM, CH-11 (18), their responses to the membrane-bound FasL have as yet been determined. We therefore cultured the SW480 cells as well as the stable clones including 480BK (vector-transfected), 480S1 and 480S2 (Tß4-overexpressing) derived from them on the formaldehyde-fixed FasL-bearing Jurkat T cells (30). As can be seen in Figure 1, Tb4 overexpressers were more resistant to the cytotoxicity of Jurkat cells than both the parental and the vector-transfected cells.


Figure 1
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  		Fig. 1. Tß4 overexpression reduces the susceptibility of SW480 cells to the toxic effects of Jurkat T cells. SW480 cells and stable clones derived from them were seeded onto 96-well plates at a density of 1 x 104/well. After 24 h, medium was replaced with the one containing 1 x 104 paraformaldehyde-fixed Jurkat cells. MTT assays were performed 48 h after coculturing. Data are mean ± SD from three independent determinations. *P < 0.05 when compared with the parental and vector-transfected SW480 cells by one-way ANOVA with repeated measures.

 
Fas expression and susceptibility to FasL in Tß4 overexpressers are restored by inhibiting MMP
Having demonstrated that Tß4 overexpressers were less sensitive to FasL-mediated toxicity, we next investigated the mechanism underlying Fas downregulation in these cells. Since Fas has recently been shown to be cleaved and inactivated by MMP-7 (31) plus increased level and activity of this protease have been demonstrated in the Tß4 overexpressers (18), we assessed whether Fas levels in these cells could be restored by suppressing MMP-7. Pretreatment of GM6001, a MMP inhibitor, indeed resulted in an increase of this death receptor not only in the 480S1 and 480S2 cells but also in the parental as well as the 480BK cells (Figure 2A). Accordingly, recovery of surface expression of Fas was also detected in both the 480BK and 480S1 cells after they were incubated with the MMP inhibitor (Figure 2B). Effect of GM6001 in altering the susceptibility of Tß4 overexpressers to CH-11 was then analyzed. As can be seen in Figure 2C, the 480S1 and 480S2 cells, after being treated with MMP inhibitor, were killed as effectively as the parental and the 480BK cells by the agonistic anti-Fas antibody.


Figure 2
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  		Fig. 2. Fas levels in the Tß4 overexpressers as well as their susceptibility to FasL are restored by MMP inhibitor. (A) Fas levels in various SW480 clones under the influence of MMP inhibitor, GM6001, were analyzed by western blotting with a polyclonal anti-Fas antibody. Equal sample loading was demonstrated by reprobing the blot with an anti-ß-tubulin antibody. (B) Surface expression of Fas in the 480BK and 480S1 cells before and after GM6001 treatment was measured by flowcytometry as described in ‘Materials and methods’. (C) Viability of various SW480 clones after CH-11 (40 ng/ml) treatment in the absence or presence of GM6001 was determined by MTT assays.

 
Tß4 overexpressers are more resistant to doxorubicin and etoposide
We next asked whether downregulated Fas expression in the Tß4 overexpressers renders them more resistant to chemotherapy since signaling via this death receptor has been shown to be responsible for the apoptosis of colon cancer cells induced by a variety of chemotherapeutic agents. Comparing with the parental and the vector-transfected SW480 cells, Tß4 overexpressers were indeed more resistant to doxorubicin and etoposide, two topoisomerase II inhibitors, but their susceptibilities to 5-FU and irinotecan remained unchanged (Table I).


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  		Table I. Susceptibility of various SW480 clones to different anticancer drugs

 
Fas/FasL signaling is not involved in the death of SW480 cells induced by doxorubicin and etoposide
Since a functional FasL/Fas pathway has previously been shown to be crucial for doxorubicin-induced apoptosis of SW480 cells (11), expression of FasL and Fas in the 480BK cells as well as in the 480S1 cells under the influence of doxorubicin and etoposide was examined by RT–PCR analysis. As can be seen in Figure 3A, neither Fas nor FasL RNA levels were increased by drug treatment. To examine the involvement of increased FasL translocation and/or Fas clustering in plasma membrane in the apoptosis triggered by doxorubicin and etoposide, cells were pre-incubated with ZB4, a neutralizing anti-Fas antibody, before drug treatment. As shown in Figure 3B, cytotoxicity of these topoisomerase II inhibitors was not affected, whereas cell death triggered by FasL (CH-11) was suppressed by the antibody. Moreover, the addition of nystatin, a lipid raft-disrupting agent that could inhibit surface clustering of Fas (32,33), did not prevent the apoptosis of these cells triggered by doxorubicin and etoposide (Figure 3C).


Figure 3
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  		Fig. 3. FasL/Fas signaling is not involved in the apoptosis of SW480 cells induced by doxorubicin and etoposide. (A) Total RNAs were isolated from both the 480BK and 480S1 cells after they were treated without or with doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) for different periods. RT–PCR analysis was subsequently performed as described in ‘Materials and methods’ using FasL-, Fas- and GAPDH-specific primer sets. (B) Cells seeded on the 96-well plates were incubated without or with 0.5 µg/ml of a Fas neutralizing antibody, ZB4, for 1 h before the addition of doxorubicin, etoposide or CH-11 (40 ng/ml). After 48 h, cell viability was determined by MTT assays. (C) Cells seeded on the 96-well plates were incubated without or with 10 µg/ml nystatin for 1 h before the addition of doxorubicin or etoposide, and 48 h later, cell viability was determined by MTT assays.

 
Expression of TRAIL, DR4 and DR5 in the SW480 cells is not induced by doxorubicin and etoposide
Since upregulation of TRAIL and/or DR4/DR5, its cognate receptors, has been reported to be responsible for drug-induced sensitization of colon cancer to this death ligand, RT–PCR analysis was performed to assess whether activation of TRAIL, DR4 and/or DR5 occurred in either the 480BK or the 480S1 cells upon doxorubicin and etoposide treatment. As can be seen in Figure 4A, RNA levels of TRAIL, DR4 and DR5 were not increased by these drugs in either cell lines. In agreement, neither were the surface levels of DR4 in the 480S1 cells stimulated by doxorubicin and etoposide (Figure 4B).


Figure 4
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  		Fig. 4. Levels of TRAIL, DR4 and DR5 are not altered in the SW480 cells treated with doxorubicin and etoposide. (A) RT–PCR analysis was carried out primarily as described in Figure 3 except TRAIL-, DR4-, DR5- and GAPDH-specific primer sets were used. (B) Flow cytometric analysis was applied to determine the surface levels of DR4 in the 480S1 cells before and after doxorubicin and etoposide treatment.

 
Caspase 9 is critical for the apoptosis of SW480 cells induced by doxorubicin and etoposide whose activation is suppressed in the Tß4 overexpressers
Since neither FasL/Fas nor TRAIL/DR4/DR5 pathway seemed to be involved in the death of SW480 cells triggered by doxorubicin and etoposide, we turned our attention to the mitochondrial pathway. As shown in Figure 5, a time-dependent activation of procaspase 9 was observed in both the 480BK and the 480S1 cells treated by the topoisomerase II inhibitors. However, the activities of caspase 9 (Figure 6A) as well as its downstream target, caspase 3 (Figure 6B), induced by these drugs were much lower in the Tß4 overexpressers. Necessity of caspase 9 in drug-triggered apoptosis of both cell lines was further indicated by a dramatic increase of their survival upon the addition of LEHD, a selective caspase 9 inhibitor (Figure 7).


Figure 5
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  		Fig. 5. Caspase 9 activation is detected in the SW480 cells treated with doxorubicin and etoposide. Total lysates (50 µg) prepared from the 480BK and 480S1 cells after being treated with doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) for varying lengths of time were subjected to western blot analysis using an anti-caspase 9 monoclonal antibody. Similar blot was reprobed with an anti-ß-tubulin antibody to show equal loading of each sample.

 

Figure 6
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  		Fig. 6. Activation of both caspases 9 and 3 induced by doxorubicin and etoposide is diminished in the Tß4 overexpressers. Total lysates (50 µg) prepared from the 480BK and 480S1 cells after being treated with doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml) for 24 h (caspase 9) or 48 h (caspase 3) were subjected to activity assays as described in ‘Materials and methods’ using LEHD-pNA and DEVD-pNA as substrates for caspase 9 (A) and caspase 3 (B), respectively.

 

Figure 7
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  		Fig. 7. Apoptosis of the SW480 cells induced by doxorubicin and etoposide is inhibited by caspase 9 inhibitor. Cells (480 BK and 480S1) seeded on the 96-well plates were incubated without or with 10 µM Z-LEHD-FMK for 12 h before the addition of doxorubicin (0.5 µg/ml) or etoposide (5 µg/ml). After 48 h, cell viability was determined by MTT assays.

 
Upregulation of Survivin is responsible for drug resistance of the Tß4 overexpressers
Since the sensitivity of the Tß4 overexpressers to doxorubicin and etoposide was dramatically decreased, involvement of several cellular apoptosis inhibitors was subsequently examined. As can be seen in Figure 8A, RNA levels of Bcl-2 and Bcl-XL, two factors critical for maintaining the integrity and preserving the functions of mitochondria during apoptotic stress (34), as well as XIAP, a potent inhibitor for caspases 9, 3 and 7 (35), were not altered in the 480S1 cells. In contrast, both RNA and protein levels of Survivin, a selective caspase 9 inhibitor (36,37), were dramatically upregulated in the Tß4 overexpressers (Figure 8). To further elucidate the role of Survivin in the increased drug resistance of these cells, we assessed the viability of both 480BK and 480S1 cells expressing less amount of the aforementioned anti-apoptotic factor to doxorubicin and etoposide. As shown in Figure 9, transient transfection of a plasmid encoding part of an antisense transcript of Survivin dramatically reduced Survivin protein levels in both cell types. Intriguingly, Survivin downregulation resulted in not only an increased sensitivity of both the 480BK and the 480S1 cells to doxorubicin and etoposide but also a restoration of the drug susceptibility of the latter to a similar level of the vector-transfected cells (Table II).


Figure 8
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  		Fig. 8. Upregulation of Survivin is detected in the Tß4 overexpressers. (A) Total RNAs isolated from four different cell lines were subjected to RT–PCR analysis primarily as described in Figure 3 except Bcl-2-, Bcl-XL-, XIAP-, Survivin- and GAPDH-specific primer sets were used. (B) Total lysates (50 µg) prepared from four different clones were subjected to western blot analysis using an anti-Survivin polyclonal antibody as a probe. Equal sample loading was demonstrated by reprobing the similar blot with an anti-ß-tubulin antibody.

 

Figure 9
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  		Fig. 9. Downregulation of Survivin in both the 480BK and the 480S1 cells by transient transfection with a plasmid encoding part of an antisense transcript of Survivin. Total lysates (50 µg) prepared from untransfected (dash), empty vector (V)- or Survivin antisense RNA-expressing plasmid (S)-transfected 480BK and 480S1 cells were subjected to western blot analysis using a polyclonal anti-Survivin antibody. Similar blot was reprobed with an anti-ß-tubulin antibody to normalize sample loading.

 

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  		Table II. Downregulation of Survivin restores the susceptibility of Tß4 overexpressers to anticancer drugs

 
Increased Survivin expression is associated with poor prognosis of patients with stage III colon cancer
Since overexpression of Survivin has been reported to be associated with shorter survival of patients with Stage II CRC after curative resection (38) as well as those who with recurrent diseases (39), we assessed whether such correlation also existed in patients with Stage III CRC. As shown in Figure 10, patients with negative Survivin IHC staining lived significantly longer than those with positive Survivin staining.


Figure 10
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  		Fig. 10. Upregulation of Survivin is associated with poor prognosis of patients with Stage III CRC. Positive (A) and negative (B) Survivin IHC staining patterns. (C) Survival curves of Stage III CRC patients according to different Survivin IHC staining patterns were plotted by Kaplan–Meier method and (closed circles) and (open circles) represent patients with positive and negative Survivin stainings, respectively (P = 0.041; log-rank test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our previous studies have shown a negative correlation between the expression levels of Tß4 and Fas in SW480 colon carcinoma cells as well as a reduced susceptibility of the Tß4-overexpressing SW480 cells (overexpressers) to the toxic effects of an agonist anti-Fas antibody, CH-11 (18). Concordantly, in the present study we found that Tß4 overexpressers (480S1 and 480S2) were killed less severely by the FasL-positive Jurkat T cells than the parental and vector-transfected (480BK) cells (Figure 1). Although downregulated Fas expression in colon cancer cells has previously been postulated by a ß-catenin-mediated disruption of nuclear factor-kappaB (NF-{kappa}B), the main transcription activator for Fas (40), our results did not support this speculation. Instead, we attributed Fas downregulation in the Tß4 overexpressers to its cleavage by MMP-7 since both total and surface levels of Fas as well as the susceptibility to CH-11 in these cells were restored by MMP inhibitor (Figure 2). In addition, only a modest decrease in NF-{kappa}B-directed reporter expression was detected in the Tß4 overexpressers (data not shown). These results together with our previous findings that both the amount and the activity of MMP-7 were increased in Tß4-overexpressing SW480 cells (18) strongly suggest a causal relationship between MMP-7 upregulation and resistance of tumor cells to FasL-mediated apoptosis (31). Thus, colon cancer cells, via Tß4 upregulation, might gain further metastatic advantages after developing the resistance to FasL-triggered apoptosis since the immune systems rely heavily on this ligand to eradicate tumors (41).

Although the FasL/Fas signaling pathway has been shown to be involved in the apoptosis of colon cancer cells induced by a variety of chemotherapeutic agents (9–11,42,43), participation of other pathways has also been reported. For example, FasL expression induced by doxorubicin, topotecan and etoposide was considered just a stress response rather than the cause of apoptosis in GL3/c1 colon carcinoma cells (44). FasL-independent activation of Fas signaling during apoptosis induced by camptothecin has been detected in HT29 colon carcinoma cells (45). In this study, we found that the Tß4-overexpressing SW480 cells were more resistant to doxorubicin and etoposide, but their susceptibilities to 5-FU and irinotecan remained unchanged (Table I). Even though increased Fas expression has previously been reported in SW480 cells incubated with 5-FU and irinotecan (9,10), the involvement of this DR in drug-induced cytotoxicity was not established. Moreover, apoptosis of HCT116 cells triggered by 5-FU, irinotecan and oxaliplatin has recently been shown to be dependent on DR5 (46), which might explain why Tß4-overexpressing SW480 cells remained sensitive to 5-FU and irinotecan because DR5 expression may not be altered by these drugs, just like it was not affected by doxorubicin and etoposide (Figure 4). While participation of the extrinsic pathways and contribution of a FasL-independent, Fas/FADD-dependent pathway reported earlier by others (47,48) in the apoptosis of SW480 cells triggered by doxorubicin and etoposide have been excluded herein (Figures 3 and 4), signaling via death receptors in similar cells initiated by the aforementioned drugs has been reported. Discrepancies between these data are currently unknown, but one possibility is that the SW480 line used by each laboratory might be different (especially the p53 status which is crucial for Fas, FasL and DR5 expression induced by DNA damage) since negligible death of SW480 cells under doxorubicin, etoposide and irinotecan treatment has also been reported (49).

In addition to the death receptor pathways, certain chemotherapeutic agents could induce the apoptosis of tumor cells by activating the mitochondrial pathway which leads to apoptosome formation and subsequent activation of caspases 9 and 3 (50). Since activation of these caspases was clearly detected in both the 480BK and the 480S1 cells incubated with doxorubicin and etoposide (Figures 5 and 6) plus apoptosis induced by them was drastically suppressed by LEHD, a selective caspase 9 inhibitor (Figure 7), we postulated that these topoisomerase II inhibitors kill SW480 cells mainly through the intrinsic pathway. Interestingly, not only were the Tß4 overexpressers more resistant to doxorubicin and etoposide than both the parental and the vector-transfected SW480 cells (Table I), but the activation levels of caspases 9 and 3 in these cells were greatly diminished (Figure 6), suggesting a decrease in the mitochondrial signaling in Tß4 overexpressers. Surprisingly, expression of neither Bcl-2 and Bcl-XL, two mitochondrial stabilizers, nor XIAP was increased in these cells (Figure 8) even though protective effects of the three aforementioned apoptosis inhibitors on SW480 cells have been reported, respectively (51–53). On the other hand, expression of Survivin (both RNA and protein), a downstream target of ß-catenin (54,55), was increased in the Tß4 overexpressers which might account for their drug resistance. This speculation was confirmed by the observation that the sensitivity to both doxorubicin and etoposide was dramatically enhanced in the Survivin-knockdown cells (Table II). However, the precise mechanism by which Survivin increases drug resistance in the SW480 cells remains to be deciphered because cleavage (activation) of procaspase 9 still occurs in the Tß4 overexpressers (Figures 5 and 6) which seems to be contradictory to the theories proposed by other investigators (36,37). Finally, a shorter survival in Stage III CRC patients with a positive Survivin staining demonstrated herein (Figure 10) reiterates the value of this anti-apoptotic factor in the prognosis for colon cancer (38,39).

In summary, we have extended our previous findings by showing that Tß4 overexpression renders the SW480 colon cancer cells more resistance to apoptosis triggered by the FasL-bearing T cells as well as two topoisomerase II inhibitors, doxorubicin and etoposide. Interestingly, the mitochondrial (intrinsic), not the death receptor (extrinsic), pathway is responsible for the death of SW480 cells induced by these drugs. Regarding the resistance mechanism of the Tß4 overexpressers to FasL and anticancer drugs, the former is likely to be accounted by MMP-7 upregulation and the latter seems to be attributed to Survivin activation. More importantly, upregulated expression of this anti-apoptotic factor is associated with a poor prognosis of the Stage III CRC patients. Taken together, our results suggest that the susceptibility of colon carcinoma cells to FasL and/or anticancer drugs (at least certain topoisomerase II inhibitors) might be increased by reducing the expression of Tß4. Additional studies are clearly warranted to determine whether such a strategy would be effective in suppressing both the in vitro and in vivo malignant progression of CRC.


   Acknowledgments
 
This study was supported by Grants NSC 93-2320-B-010-047 and NSC 94-2320-B-010-050 from the Nation Science Council of the Republic of China. Funding to pay the Open Access publication charges for this article was provided by the National Science Council of Republic of China.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 18, 2005; revised December 11, 2005; accepted December 13, 2005.


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