Main

MRTs were first described in the kidney as a rare variant of Wilms' tumor with a rhabdomyosarcomatoid pattern. Unfortunately, there is no effective treatment for this disease because it is largely resistant to both chemotherapy and radiotherapy. MRT also has an extremely poor prognosis because of its high potential for metastasis (1). Primary MRT has been described in the CNS, pelvis, and paravertebral regions (2–7). A characteristic feature of MRT cells is the presence of a large eosinophilic inclusion in the cytoplasm (8, 9). In the brain, the tumors may present as a mixture of rhabdoid tumor with primitive neuroectodermal tumors, mesenchymal, or epithelial element, an entity referred to as atypical teratoid/rhabdoid tumors (10, 11). Most atypical teratoid/rhabdoid tumors demonstrate monosomy 22 or deletions of chromosome band 22q11 with alterations of the hSNF5/INI1 gene (12). Recently study revealed that MRT has truncated mutations and homozygous deletions of hSNF5/INI1 gene, suggesting a tumor suppressor gene (13).

TNF-related apoptosis-inducing ligand or Apo2 ligand (TRAIL/Apo2L) was recently identified as a member of the TNF superfamily of cell death-inducing ligands. This ligand is a type II transmembrane protein that triggers apoptosis mainly in tumor cells (14, 15). TRAIL induces apoptotic cell death in many cancer or transformed cells both in vitro and in various in vivo tumor models (16–20). However, its proapoptotic effects are minimal in normal human cells in vitro and in TRAIL-treated animals (14, 21–25). TRAIL mRNA is constitutively present in many tissues, unlike the restricted expression of other proapoptotic members of the TNF family (21, 22). This feature of TRAIL/Apo2L prompted us to investigate whether this cell death ligand is active against MRT cells.

TRAIL/Apo2L has four different specific receptors: two proapoptotic death receptors (DR4, DR5), and two antiapoptotic decoy receptors (DcR1, DcR2). Normal cells express all these receptors. The preferential expression of these decoy receptors in normal tissues led to the hypothesis that both molecules protect normal tissues from TRAIL-induced apoptosis by competing with the DR4 and DR5 receptors for limited amounts of ligand (22, 26–28). The increased TRAIL sensitivity of tumor cells has been postulated to result from the lack of DcR1 or DcR2 expression, and recently study revealed that DcR1 and DcR2 expressions were down-regulated in neuroblastoma and primitive neuroectodermal tumor cell lines (29).

Doxorubicin had been reported to enhance the apoptotic activity of TRAIL and to up-regulate DR5 receptor expression in MCF-7 breast cancer cell line (30) and multiple myeloma cells (31).

NF-κB is an important part of cell survival pathways and confers protection of tumor cells against various proapoptotic agents (i.e. TNFα) (32, 33). The cell-permeable peptide SN50 binds to the nuclear localization sequence of NF-κB, blocks its nuclear translocation, and inhibits its transcriptional activity (34).

On cancer cell proliferation, the PI3-kinase/Akt pathway is also important. Akt is activated in response to activation by many different growth factors (i.e. platelet-derived growth factor) (35), and inhibits apoptotic cell death in cancer cells. Once activated, Akt exerts antiapoptotic effects through phosphorylation of substrates such as Bad (36) and caspase-9 (37), and activates the antiapoptotic NF-κB-mediated transcriptional pathway (38, 39).

The aim of this study was to examine whether TRAIL/Apo2L can induce apoptosis in human MRT cell lines and to investigate the difference between TRAIL receptor expressions in TRAIL-sensitive versus TRAIL-resistant MRT cells. Moreover, we wished to examine whether doxorubicin, NF-κB inhibitor (SN50), and PI3-kinase/Akt inhibitor (wortmannin, LY294002) can enhance the activity of TRAIL/Apo2L ligands in these cells. Our results suggest the possibility for use of TRAIL/Apo2L as a novel therapeutic agent for MRT treatment.

METHODS

Cell Culture

The MRT cell lines used in this study (TM87-16, STM91-01, TTC642, TTC549, and TTC1240) were provided by Dr. Hiroyuki Shimada and Dr. Timothy J. Triche (Childrens Hospital Los Angeles, Los Angeles, CA, U.S.A.). The MRT cell line YAM-RTK1 was provided by Dr. Kanji Sugita (Yamanashi Medical University, Kofu, Japan). To investigate these cells in our study, informed consent was obtained from those laboratories. Diagnosis of these MRT cell lines was based on finding the presence of a large eosinophilic inclusion in the cytoplasm by light microscopy, and it was recently reported that these cell lines have homozygous deletion (TM87-16, TTC 549, STM91-01, YAM-RTK1) and point mutations (TTC642, TTC1240) of the hSNF5/INI1 gene (40). Ewing's sarcoma cell line ES-1OT was established from primary tumors in our laboratory. HeLa cells were used as a positive control for DR4/DR5 expression. The MRT and Ewing's sarcoma cell lines used in this study were isolated from the 16th through 23rd passages. The cells were cultured in RPMI 1640 (GIBCO, Gaithersburg, MD, U.S.A.) supplemented with 2.5% to 5% FCS (ICN Biomedicals Inc, Cleveland, OH, U.S.A.).

Reagents

Antibodies against DR4, DR5, DcR1, and β-actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, U.S.A.). Anti-DcR2 and human-recombinant TRAIL, the inhibitory peptide SN50, an inactive control for SN50 peptide (SN50M), wortmannin, and LY294002 were from Calbiochem (San Diego, CA, U.S.A.). Doxorubicin was from Sigma Chemical Co (St. Louis, MO, U.S.A.). Enhanced chemiluminescence Western blot detection reagents, which include the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, were from Amersham Pharmacia Biotech Inc (Arlington Heights, IL, U.S.A.); goat anti-rabbit IgG (FITC)-conjugated F(ab′)2 fragment (Organon Teknika Corp, West Chester, PA, U.S.A.), rat anti-mouse IgG (FITC)-conjugated F(ab′)2 fragment, and rat anti-goat IgG (FITC)-conjugated F(ab′)2 fragment (Vector Laboratories, Inc, Burlingame, CA, U.S.A.) were used for immunofluorescence examination. Coomassie protein assay reagent kit was from Pierce, Inc (Rockford, IL, U.S.A.). The annexin V-PI early apoptosis detection kit was from Medical & Biologic Laboratories Co, LTD (Nagoya, Japan). PCR kits for DNA ladder assays were obtained from Maxim Biotech, Inc (San Francisco, CA, U.S.A.), and MTT was obtained from Nachalai tesque (Kyoto, Japan).

Western Blot Analysis

Six MRT cell lines and one Ewing's sarcoma cell line were examined by Western blot analysis for the expression of the DR4, DR5, DcR1, and DcR2 TRAIL/Apo2L receptors. For the doxorubicin experiments, MRT cells were preincubated with doxorubicin (250 or 500 ng/mL) for 24 h. Protein concentrations were measured using Coomassie protein assay reagent kit (Pierce, Inc). The protein samples were subjected to 7.5% SDS-PAGE and transferred onto polyvinylidine difloride (PDVF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA). The membranes were blocked with 3% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 and incubated with an anti-DR4, -DR5, -DcR1, or -DcR2 antibody and then with horseradish-peroxidase-conjugated secondary antibodies. The immunoblots were visualized using enhanced chemiluminescence detection (enhanced chemiluminescence plus detection reagent; Amersham Pharmacia Biotech).

Detection of TRAIL Receptor by Flow Cytometric Analysis

The MRT cell lines were characterized for their surface receptor expression by flow cytometry. HeLa cells served as a positive control for DR4/DR5 expression and as a negative control for DcR1 and DcR2 expression. MRT cells (1 × 106) were preincubated with doxorubicin (250 or 500 ng/mL) for 24 h and incubated with each anti-TRAIL receptor antibody or a respective control for 1 h. DcR1 and DR5 expressions were assessed with anti-DcR1 and anti-DR5 MAb and confirmed with goat anti-human DcR1 and DR5 polyclonal antibodies. DR4 expression was assessed with anti-DR4 MAb and mouse anti-DR4 MAb. DcR2 expression was assessed with anti-DcR2 MAb and a rabbit anti-DcR2 polyclonal antibody. Cells were washed with PBS and incubated for 1 h with rat anti-goat IgG FITC-conjugated F(ab′)2 fragment for anti-DcR1 and anti-DR5 MAb expression, with a rat anti-mouse IgG FITC-conjugated F(ab′)2 fragment for DR4 MAb expression, or with an anti-rabbit IgG FITC-conjugated F(ab′)2 fragment for DcR2 MAb expression. Cells were washed with PBS and analyzed by flow cytometry with a fluorescence-activated cell sorting scan using CELLQuest software (Becton-Dickinson, Mountain View, CA, U.S.A.).

Survival, Death, and Apoptosis Assays

MTT colorimetric assay.

MRT or Ewing's sarcoma cells (2 × 104) were plated in a 96-well microplate and incubated for 18 h with TRAIL (10–1000 ng/mL) in 2.5–5% bovine serum-supplemented RPMI medium. In some experiments, MRT cells were pretreated with doxorubicin (250 or 500 ng/mL for 4 h), the NF-κB inhibitory peptide (SN50, SN50M) at 30 ng/mL for 3 h, or PI3-kinase/Akt pathway inhibitor (200 nM wortmannin, 20 μM LY294002 for 4 h) before exposure to TRAIL. At the end of treatment, cells were incubated with 1 mg/mL MTT for 4 h at 37°C. A mixture of isopropanol and 1 N HCl (24:1, vol/vol) was added with pipetting to dissolve the formazan crystals. Dye absorbance in viable cells was measured at 595 nm, with 670 nm used as a reference wavelength.

Annexin V/PI staining.

Detection of early apoptotic cells was performed with an MEBOCYTO apoptosis kit (Annexin V-FITC kit, Medical & Biologic Laboratories Co, LTD). Cells (2 × 105) were exposed for 4 h to TRAIL or media, washed with PBS, resuspended in binding buffer, incubated in the dark at room temperature with annexin V-FITC and PI for 15 min, and analyzed by dual-color flow cytometry. Cells that were annexin V-FITC+, with translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, and PI−, with intact cellular membranes, were considered to be early apoptotic cells.

DNA ladder assay.

For the detection of nucleosomal ladders in apoptotic cells, a DNA Ladder Assay PCR kit (Maxim Biotech, Inc) was used. Cells (1 × 106) were incubated with 10–1000 ng/mL TRAIL for 24 h, and genomic DNA was then extracted from the cells by Maxim's DNA isolation kit (Maxim Biotech, Inc). Adaptor-ligation of DNA was performed at 16°C for 16 h, and PCR was then initiated using the adaptor-primer. Aliquots of PCR products were electrophoresed through 2% agarose gels containing 0.2 mg/mL ethidium bromide.

RESULTS

TRAIL/Apo2L-induced apoptosis in MRT cell lines.

In three of the six MRT cell lines tested (TM87-16, TTC642, and STM91-01) and in the ES-1OT cell line, TRAIL/Apo2L induced significant reduction of cell survival as assessed by the MTT colorimetric assay (Fig. 1A). The survival rate of STM91-01 and ES-1OT cells was more than 70% after treatment with 100 ng/mL of TRAIL. In contrast, TM87-16 and TTC642 cells showed less than 40% survival at the same dose, so these lines were considered TRAIL-sensitive. Three cell lines (TTC549, TTC1240, and YAM-RTK1) showed more than 80% survival even after exposure to a high dose (1000 ng/mL) of TRAIL/Apo2L. So these were considered to be TRAIL-resistant (Fig. 1B).

Figure 1
figure 1

MTT colorimetric assay of MRT cells cultured with human recombinant TRAIL. Dose-response survival curves were generated for MRT cells treated with TRAIL (10–1000 ng/mL) for 18 h. A, survival curves for TRAIL-sensitive MRT cell lines: TM87-16, TTC642, STM91-01, and ES-1OT. B, survival curves for TRAIL-resistant MRT cell lines: TTC549, TTC1240, and YAM-RTK1.

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Apoptosis in TRAIL-sensitive or -resistant MRT cells was confirmed by annexin V-FITC/PI staining: early apoptotic cell death was identified with annexin V+PI− staining, and late apoptotic cell death and necrosis were identified with annexin V+PI+ staining. In contrast with TRAIL-untreated cells (Fig. 2A), TRAIL-induced apoptotic cell death occurred after 4 h (Fig. 2B).

Figure 2
figure 2

Annexin V/PI staining of MRT cells cultured with human recombinant TRAIL. MRT cells were cultured for 4 h with TRAIL (250 ng/mL). A, TRAIL-untreated cells. B, significant induction of apoptotic cell death (annexin V+PI− status) detected in the TTC642 cell line.

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We also examined whether TRAIL-induced, dose-dependent apoptotic cell death occurs in MRT cell lines by PCR-DNA ladder assay. TM87-16 cells were incubated with increasing concentrations (0, 10, 50, 100, and 1000 ng/mL) of TRAIL/Apo2L for 4 h. As the concentration of TRAIL/Apo2L increased, the production of DNA ladder fragments was also increased (Fig. 3). However, TRAIL-resistant cell lines did not have increased DNA ladder fragments after treatment with TRAIL (data not shown).

Figure 3
figure 3

Detection of apoptotic DNA in the TM87-16 cell line cultured with TRAIL. TM87-16 MRT cells were cultured for 4 h with TRAIL (0–1000 ng/mL). Lane 1, control, cells cultured with 0 ng/mL; lane 2, cultured with 10 ng/mL TRAIL; lane 3, cultured with 50 ng/mL TRAIL; lane 4, cultured with 100 ng/mL TRAIL; and lane 5, cultured with 1000 ng/mL TRAIL.

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TRAIL receptor expression in MRT cell lines.

We investigated how TRAIL/Apo2L sensitivity of MRT cells correlates with the expression of TRAIL receptors using immunoblotting method. Western blot analysis of six MRT cell lines (TM87-16, TTC642, TTC549, STM91-01, YAM-RTK1, and TTC1240) revealed expression of all four receptors (DR4, DR5, DcR1, and DcR2). In all MRT cell lines, the levels of DR4 and DR5 expression were consistently detectable, whereas the expression of DcR1 and DcR2 was variable. In contrast with TRAIL-resistant cells, the expression of DcR2 was low in TRAIL-sensitive cell lines (TM87-16, TTC642, and STM91-01). DcR1 expression levels were not different in TRAIL-sensitive versus TRAIL-resistant MRT cells (Fig. 4).

Figure 4
figure 4

Detection of TRAIL receptor expression by Western blot analysis. Western blot analysis for DR4, DR5, DcR1, and DcR2 receptors was performed in MRT cells. ES-1OT (Ewing's sarcoma cell line) and HeLa cell were used as a positive control.

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Doxorubicin induces a significant up-regulation of TRAIL receptors.

Next, we examined whether the genotoxic agent doxorubicin can cause an increase in the expression levels of TRAIL receptors in MRT cell lines. TM87-16 MRT cell line was treated with doxorubicin (250 or 500 ng/mL) for 24 h, and TRAIL receptor expression was analyzed using Western blot and flow cytometric analyses. After treatment with doxorubicin, DR4 and DcR2 expression was somewhat up-regulated, whereas DR5 was significantly up-regulated (Fig. 5A). On the contrary, after treatment with doxorubicin, flow cytometric analysis revealed that surface expression of DcR1 was not up-regulated, whereas expression of DR5 increased (Fig. 5B). The same results were observed in the other five MRT cell lines (data not shown).

Figure 5
figure 5

Doxorubicin (Doxo) up-regulates DR5 receptor expression in the TM87-16 cell line. A, immunoblotting analysis indicates significant dose-dependent up-regulation of DR5 expression by doxorubicin treatment (250 or 500 ng/mL). Doxorubicin also induces up-regulation of DR4 expression. B, flow cytometric analysis shows significant up-regulation of surface DR5 expression by doxorubicin treatment (500 ng/mL). However, surface expression of DcR1 is not overexpressed by pretreatment with doxorubicin.

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Doxorubicin enhances induction of apoptotic cell death by TRAIL receptors in MRT cell lines.

We investigated whether doxorubicin could enhance the proapoptotic effect of TRAIL in TRAIL-sensitive MRT cell lines. TM87-16 and TTC642 cell lines, TRAIL-sensitive MRT cells, were pretreated with doxorubicin (250 or 500 ng/mL) for 4 h and then incubated with TRAIL/Apo2L (100 ng/mL) for 18 h. MTT colorimetric assay revealed that doxorubicin significantly enhanced the proapoptotic effect of TRAIL/Apo2L in these cell lines, reducing their survival rate to levels lower than those expected. Moreover, doxorubicin enhanced the susceptibility to TRAIL in YAM-RTK1, TRAIL-resistant MRT cell. Doxorubicin and TRAIL/Apo2L therefore appear to have a synergistic interaction in promoting apoptosis (Fig. 6).

Figure 6
figure 6

Doxorubicin (Doxo) enhances the sensitivity to TRAIL in TM87-16, TTC 642, and YAM-RTK1 cell lines. MTT colorimetric assay of TM87-16 and TTC642 (TRAIL-sensitive) cells and YAM-RTK1 (TRAIL-resistant) cells cultured with doxorubicin (250, 500 ng/mL) or human recombinant TRAIL (100 ng/mL). Doxorubicin and TRAIL together significantly reduce the survival rate of TM87-16 and TTC642 cells. Pretreatment of doxorubicin enhances the susceptibility to TRAIL in YAM-RTK1.

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NF-κB inhibition or PI3-kinase inhibition modulates the effect of TRAIL on MRT cells.

We examined whether inhibition of NF-κB transcriptional activity could modulate the response of MRT cells to TRAIL. TTC642, TRAIL-sensitive MRT cell, STM91-01, moderately TRAIL-sensitive cell, and YAM-RTK1 and TTC549, TRAIL-resistant cells, were preincubated for 4 h with the cell-permeable peptide SN50 at 30 ng/mL, a nontoxic concentration 31), and then incubated with TRAIL/Apo2L (50 ng/mL) for 18 h. Pretreatment with SN50 enhanced the sensitivity of each MRT cell line to TRAIL (Fig. 7), whereas an inactive control for SN50 peptide, SN50M, did not reduce the survival rate in spite of TRAIL (data not shown).

Figure 7
figure 7

NF-κB inhibitor SN50 with TRAIL pretreatment increased the number of apoptotic cell deaths in TTC642, STM91-01, YAM-RTK1, and TTC549 cell lines. MTT assay of TTC642 and STM91-01 (TRAIL-sensitive) MRT cells and YAM-RTK1 and TTC549 (TRAIL-resistant) MRT cells cultured with NF-κB (30 ng/mL, nontoxic dose) or human recombinant TRAIL (100 ng/mL). NF-κB inhibitor and TRAIL together reduce the survival rate of TRAIL-sensitive and -resistant MRT cells.

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We also investigated whether PI3-kinase/Akt inhibitor sensitized MRT cells to TRAIL-induced apoptosis. TTC642 and YAM-RTK1 were pretreated with wortmannin (200 nM) or LY294002 (20 μM) for 4 h and then TRAIL (50 ng/mL) was added for 18 h. Wortmannin/LY294002 pretreatment significantly augmented the sensitivity of TRAIL-sensitive MRT cells to TRAIL, and the pretreatment of PI3-kinase inhibitor also enhanced the sensitivity of TRAIL-resistant MRT cells (Fig. 8).

Figure 8
figure 8

PI3-kinase inhibitor enhanced the susceptibility to TRAIL in MRT cell lines. MTT assay of MRT cells cultured with PI3-kinase inhibitor (200 nM wortmannin and 20 μM LY294002) or human recombinant TRAIL (50 ng/mL). Wortmannin/LY294002 and TRAIL together significantly increased the apoptotic cell death of TTC642. STM91-01 cells exhibited the same result (data not shown). Pretreatment with wortmannin/LY294002 also reduced the survival rate of the TRAIL-resistant YAM-RTK1 cell line.

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DISCUSSION

MRTs are basically resistant to current chemotherapeutic agents, radiotherapy, and stem cell transplantation, and therefore have an extremely poor prognosis. Members of the TNF family (TNF, Fas ligand, and TRAIL) have been shown to be potent inducers of apoptotic cell death in various cancer cell lines. TNF and Fas ligand are toxic to both cancer tissue and normal tissue, whereas TRAIL exerts potent antitumor activity in vitro without exhibiting systemic toxicity. It has been reported that TRAIL can induce apoptosis in some neoplasias (41–44), and a recent study revealed that TRAIL is hepatotoxic in cholestasis in vivo(45). This prompted us to examine whether TRAIL/Apo2L can induce apoptosis in MRT cells. In this study, we found that apoptosis can be induced by TRAIL/Apo2L in some MRT cell lines, and our results demonstrate that MRT cell lines can either be TRAIL/Apo2L-sensitive or TRAIL/Apo2L-resistant.

The Western blot analysis of TRAIL receptor expression in MRT cell lines was designed to identify what confers TRAIL sensitivity to MRT cells. Decoy receptors do bind ligand; they inhibit the effects of ligand binding or effective signaling. Although the death signal of DR4 and DR5 receptor is expressed in all MRT cell lines, Western blot analysis revealed that DR5 expression was variable in all of the cell lines. The expression levels of DR4 and DR5 did not correlate with TRAIL sensitivity or resistance in MRT cell lines, which is consistent with some previous observations in various tumors (30, 46, 47) but not with others (48). A previous study has reported that glioma cells have an increased level of expression for the DR5 death receptor (44). It has also been described that other neoplasias have mutations in the DR5 receptor death domain (49, 50). It is possible that our TRAIL-resistant MRT cell lines might also have mutations in this domain.

It has been suggested that DcR1 and DcR2 expression may provide a mechanism for cells to avoid TRAIL/Apo2L-induced apoptosis; therefore, we investigated whether expression of these decoy receptors correlate with TRAIL resistance. We found that DcR1 and DcR2 are expressed in all MRT cell lines, particularly in the TRAIL-resistant cell lines TTC549 and TTC1240. We also found that the expression level of DcR2 was low in TRAIL-sensitive MRT cell lines. The same results were reported recently (29), and some reports suggested that the expression levels of decoy receptor were not related to TRAIL sensitivity in various cancer cells (25, 31, 51, 52). Our findings suggest that there is correlation between TRAIL decoy receptor expression and susceptibility to TRAIL-induced apoptosis in MRT cell lines.

Our results could be explained by the presence of cell death inhibitors in MRT cells. FLIP [FLICE (Fas-associated death domain-like IL-1β-converting enzyme) -inhibitory protein], a caspase inhibitor, may play a role in TRAIL-induced apoptotic cell death: some investigators have reported that FLIP could be a predictor of TRAIL sensitivity (53), although others have not been able to show such a correlation (48). Other investigators recently reported that in neuroblastoma cell lines, resistance to TRAIL-induced apoptotic cell death correlated with the loss of caspase-8 expression (54). Downstream intracellular modulators of apoptosis may therefore regulate susceptibility to TRAIL/Apo2L-induced apoptosis in MRT cells. However, the mechanism underlying the resistance in TRAIL-induced apoptotic cell death remains to be elucidated.

Because doxorubicin has been reported to up-regulate expression of the DR5 death signaling receptor (54–56), we investigated whether it could sensitize MRT cells to TRAIL/Apo2L. We found that in MRT cell lines, doxorubicin somewhat induces the up-regulation of DR4 and DcR2 expression and significantly induces up-regulation of DR5 expression, as shown by Western blot and flow cytometric analyses. Previous reports have described similar findings in other malignancies (55–57). We have also demonstrated that pretreatment with doxorubicin at clinically relevant concentrations (31) significantly enhances the TRAIL sensitivity of MRT cells. Furthermore, doxorubicin enhanced the susceptibility to TRAIL in TRAIL-resistant MRT cells. Doxorubicin and TRAIL may synergistically induce apoptotic cell death via the up-regulation of TRAIL receptor expression.

TRAIL/Apo2L binding to DR4 and DR5, as well as to decoy receptor DcR2, activates NF-κB by receptor-interacting protein (RIP)-mediated activation of IκB kinase (58–60). Previous reports informed that the constitutive activation of NF-κB prevented TRAIL-induced apoptosis and inhibition of NF-κB activity enhanced the susceptibility to TRAIL in renal carcinoma cells or breast cancer cells (61, 62). However, NF-κB activity in MRT cells still remains unknown. Therefore, we investigated whether SN50, which inhibits NF-κB, could also sensitize MRT cells to TRAIL/Apo2L. Pretreatment with SN50 enhanced the sensitivity to TRAIL in TRAIL-sensitive cell lines, whereas SN50 reversed the TRAIL resistance of TTC549, YAM-RTK1, and TTC1240 MRT cells. These data demonstrate that modulation of NF-κB activation is a key determinant of the sensitivity of MRT cells to TRAIL-induced apoptosis.

PI3-kinase/Akt also plays an important role in cell survival pathways. Recent studies have shown that constitutively active Akt in tumor cells can inhibit the ability of TRAIL-inducing apoptosis (63) and that TRAIL-resistant malignant cells lack active lipid phosphatase PTEN (phosphatase and tension homologue deleted on chromosome 10, a negative regulator of the PI3-kinase/Akt pathway (64). However, the effects of specific inhibition of PI3-kinase/Akt activity in MRT cells still remain unknown. We investigated whether the PI3-kinase/Akt signaling pathway could modulate the sensitivity of MRT cells to TRAIL in MRT cell lines. PI3-K inhibitors (wortmannin, LY294002) enhanced the susceptibility to TRAIL/Apo2L in TRAIL-sensitive MRT cells. And these PI3-K inhibitors with TRAIL reduced the survival rate of TRAIL-resistant MRT cells. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to induce cell death in MRT cells.

CONCLUSIONS

In summary, although TRAIL/Apo2L is a potent inducer of apoptosis in MRT cells, sensitivity of these cells to TRAIL/Apo2L is not correlated with the expression levels of DR4 and DR5, whereas the expression of decoy receptors DcR1 and DcR2 may have correlation with the susceptibility to TRAIL. Doxorubicin enhances the expression levels of DR4 and DcR2 and significantly up-regulates the expression levels of DR5. Doxorubicin also enhances the sensitivity of MRT cell lines to TRAIL/Apo2L. Moreover, NF-κB inhibition and PI3-kinase/Akt signaling inhibition enhanced the sensitivity of these cells to TRAIL. These studies may provide a basis for clinical use of TRAIL/Apo2L for the treatment of MRT, but further testing is required in xenografts or other animal models, before use in humans.