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Overexpression of Bcl2 Blocks TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis in Human Lung Cancer Cells
Biochemical and Biophysical Research Communications 280, 788 –797 (2001) doi:10.1006/bbrc.2000.4218, available online at http://www.idealibrary.com on
Overexpression of Bcl2 Blocks TNF-Related ApoptosisInducing Ligand (TRAIL)-Induced Apoptosis in Human Lung Cancer Cells Shi-Yong Sun,* Ping Yue,* Jun-Ying Zhou,† Yinghong Wang,† Hyeong-Reh Choi Kim,† Reuben Lotan,* and Gen Sheng Wu† ,1 *Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030; and †Program in Developmental Therapeutics, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201
Received December 20, 2000
The tumor necrosis factor (TNF) related apoptosisinducing ligand (TRAIL or Apo2L) and its receptors are members of the tumor necrosis factor superfamily. TRAIL triggers apoptosis by binding to its two proapoptotic receptors DR4 and DR5, a process which is negatively regulated by binding of TRAIL to its two decoy receptors TRID and TRUNDD. Here, we show that TRAIL effectively induces apoptosis in H460 human non-small-cell lung carcinoma cells via cleavage of caspases 8, 9, 7, 3, and BID, release of cytochrome c from the mitochondria, and cleavage of poly (ADPribose) polymerase (PARP). However, overexpression of Bcl2 blocked TRAIL-induced apoptosis in H460 cells, which correlated with the Bcl2 protein levels. Importantly, the release of cytochrome c and cleavage of caspase 7 triggered by TRAIL were considerably blocked in Bcl2 overexpressing cells as compared to vector control cells. Moreover, inhibition of TRAILmediated cytochrome c release and caspase 7 activation by Bcl2 correlated with the inability of PARP to be cleaved and the inability of the Bcl2 transfectants to undergo apoptosis. Thus, these results suggest that Bcl2 can serve an anti-apoptotic function during TRAIL-dependent apoptosis by inhibiting the release of cytochrome c and activation of caspase 7, thereby blocking caspase 7-dependent cleavage of cellular substrates. © 2001 Academic Press Key Words: TRAIL; Bcl2; caspases; apoptosis; mitochondria.
Programmed cell death or apoptosis is a genetically controlled mechanism essential for the maintenance of tissue homeostasis, proper development and the elimination of unwanted cells. In mammalian cells, two 1
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major apoptosis pathways are proposed: the first one involves signals transduced through death receptors; the second relies on a signal from the mitochondria (1, 2). Both pathways are involved in an ordered activation of a set of cysteine proteases called caspases (3), which in turn cleave cellular substrates and result in the morphological and biochemical changes characteristic of apoptosis (4, 5). In the death receptor pathway, apoptosis induction involves the engagement of a set of ligands and their corresponding receptors and then transmission of the apoptotic signal in the cytoplasm by a number of caspases (6, 7). There are a number of ligand-receptor systems and target caspases involved in apoptosis. These death-inducing receptors belong to the tumor necrosis factor (TNF) superfamily including Fas/APO1, TNFR, and TRAIL receptors, all of which are characterized by a cysteine-rich extracellular domain and an intracellular “death domain” (7). When a ligand binds to the receptor, the receptor becomes trimerized and activated. The activated receptor recruits a death domain containing adaptor and a pro-caspase 8 molecule through protein-protein interactions to form a deathinducing signaling complex (DISC) (8). FADD is directly recruited into the DISC during Fas-mediated apoptosis, while TNF-induced apoptosis involves both FADD and TRADD (7). Recruitment of pro-caspase 8 to the DISC activates caspase 8 by oligomerizationinduced autocatalytic processing (9), which results in activation of downstream caspases, such as caspases 3, 6, and 7, and induction of apoptosis (7). TRAIL, a recently identified member of the TNF family, has been shown to induce apoptosis in a variety of transformed or tumor cells but not normal cells (10, 11). To date, five human TRAIL receptors have been identified; they are DR4 (12), DR5 (KILLER, TRAIL-R1, TRICK2) (13–17), TRID (TRAIL-R3, DcR1, or LIT) (15, 18 –20), TRUNDD
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(DcR2 or TRAIL-R4) (21–23), and osteoprotegerin (24). Additionally, a mouse TRAIL receptor has been identified (25). Both human DR4 and DR5 are proapoptotic receptors and binding of TRAIL to these receptors leads to the recruitment of the adapter and induction of apoptosis through activation of caspases (7). However, unlike DR4 and DR5, TRID lacks an intracellular domain and TRUNDD has a truncated death domain. Thus, these two receptors act as decoy receptors to antagonize TRAIL-induced apoptosis by competing for ligand binding (7). The mechanism of TRAIL receptormediated apoptosis is not completely understood. A study from FADD knockout cells showed that FADD is not required for TRAIL-mediated apoptosis (26). However, recent reports suggested that TRAIL-mediated apoptosis involves activation of FADD and caspase 8 (27–29). In contrast to the death receptor pathway, apoptosis can also be triggered by the mitochondrial pathway. In response to apoptotic stimuli, such as DNA damage (including anticancer drug treatment and growth factor withdrawal), damaged mitochondria release cytochrome c into the cytosol where cytochrome c can associate with Apaf1 and procaspase 9 in the presence of dATP, leading to activation of caspase 9 (30). Subsequently, the activated caspase 9 cleaves downstream caspases, such as caspases 3, 6, and 7, leading to cell death (31). In addition, activation of caspase 8 through engagement of death receptors can also trigger the mitochondrial pathway via Bid, a pro-apoptotic member of the Bcl2 family. This activation of the mitochondrial pathway is believed to amplify death receptorinduced apoptosis. Because mitochondrial damage and subsequent cytochrome c release are critical in apoptosis induction in response to some death stimuli, studies have been carried out to elucidate the role of members of the Bcl2 family in regulating mitochondrial integrity. The Bcl2 family contains anti-apoptotic members, such as Bcl2 and Bcl-X L, and pro-apoptotic members, such as Bax and Bid. It is believed that the ratios of proapoptotic and anti-proapoptotic members determine whether cells live or die (32). Both Bcl2 and Bcl-X L are localized on the cytoplasmic side of the mitochondrial membrane, the endoplasmic reticulum, and nuclear membrane (33). It has been shown that overexpression of Bcl2/Bcl-X L can protect cells from death induced by a variety of agents, such as UV irradiation, cytotoxic drugs, growth factor withdrawal, p53, and c-myc overexpression. In addition, Bcl2/ Bcl-X L can block the release of cytochrome c and thus inhibit apoptosis (34 –36). Lastly, Bcl2 can protect some cell types from Fas and TNFR1-mediated apoptosis (37–39). In this study, we investigated the role of Bcl2 in regulating TRAIL-mediated apoptosis. Here we report that TRAIL can induce apoptosis involving a caspase
cascade including activation of caspases 8, 9, 3, 7, cleavage of Bid, release of cytochrome c from mitochondria, and cleavage of PARP in H460 cells. More importantly, we show that overexpression of Bcl2 protects H460 cells from TRAIL-induced apoptosis. Furthermore, we show that Bcl2 inhibits cytochrome c release, caspase 7 activation, and PARP cleavage induced by TRAIL. Our results suggest that if TRAIL were applied for human cancer therapy, tumors that overexpress the Bcl2 protein may not be sensitive to TRAIL-induced cell death. MATERIALS AND METHODS Cells, cell culture, and transfection conditions. Seven human NSCLC cell lines (H292, H460, H1944, H522, H1792, H596, and H157) were obtained from Dr. Adi Gazdar (University of Texas Southwestern Medical Center, Dallas, TX). Three additional lines (SK-MES-1, A549 and Calu-1) were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in a 1:1 (v/v) mixture of DMEM and Ham’s F12 medium supplemented with 5% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. To generate cell lines in which the Bcl2 gene is overexpressed, H460 cells were transfected with either pSFFV-neo, an empty vector, or pSFFVneo-HA Bcl2, a hemagglutinin-tagged full length Bcl2 expression vector (a gift from Lawrence Boise, University of Miami, Miami, FL) as previously described (44). Following 2 weeks of selection in the presence of 500 g/ml of G418 (GIBCO/BRL, Rockville, MD), individual clones were isolated as described previously (57). Clones that expressed no exogenous Bcl2 (vector control) or different levels of exogenous Bcl2 protein determined by Western blot analysis were used for this study. Cell survival assay. Cells were seeded at densities of 1 ⫻ 10 4 cells per well in 96-well tissue culture plates. Cells were treated with different concentrations of TRAIL. After treatment for the indicated times, cell numbers were estimated by the sulforhodamine B (SRB) assay as described previously (58). Cell survival was calculated by using the equation: % cell survival ⫽ (A t/A c) ⫻ 100, where A t and A c represent the absorbance in treated and control cultures, respectively. DNA fragmentation assay. Cells were plated at densities of 5 ⫻ 10 3 cells per well in 96-well cell culture plates 1 day before treatment. After a 24 h-treatment, DNA fragmentation was evaluated by examination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) using a Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions. Western blot analysis. Whole cell lysates from both attached and detached floating cells were prepared as described previously (46) and protein concentration was determined using the Protein Assay Kit (Bio-Rad, Hercules, CA). Cell lysates (50 g) were electrophoresed through 7.5–12% denaturing polyacrylamide slab gels and transferred to a PROTRAN nitrocellulose transfer and immobilization membrane (Schleicher & Schuell, Inc., Keene, NH) by electroblotting. The blots were probed or reprobed with the antibodies, and then antibody binding was detected using the Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products, Boston, MA) according to the manufacturer’s protocol. Mouse anticaspase-3 (clone 19) and mouse monoclonal anti-caspase-7 (clone B94-1) antibodies were purchased from PharMingen (San Diego, CA). Rabbit polyclonal anti-caspase-9 antibody was purchased from New England BioLabs, Inc. (Beverly, MA). Goat polyclonal anticaspase-8 (C-20) and rabbit polyclonal anti-caspase-10 (H131) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa
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Cruz, CA). Rabbit polyclonal anti-Bid antibody was purchased from Trevigen (Gaithersburg, MD). Rabbit polyclonal anti-PARP (VIC 5) and anti-actin antibodies were purchased from Roche Molecular Biochemicals and Sigma Chemical Co. (St. Louis, MO), respectively. Measurement of cytochrome c release. Cells were plated onto 10-cm diameter dishes one day before treatment. After the cells were exposed to TRAIL for the indicated times, both floating and attached cells were harvested and washed once with PBS. The cell pellets were resuspended in buffer containing 0.25 M sucrose, 30 mM TrisHCl, pH 7.9, 1 mM EDTA and protease inhibitor cocktail (PharMingen) and homogenized with a glass dounce homogenizer and a B pestle (30 strokes). After centrifugation at 14,000 rpm for 15 min, the supernatants were transferred to another ultracentrifuge tube and centrifuged at 100,000 g for 30 min. The supernatants were then collected and stored at ⫺80°C. Protein concentration was determined with the Protein Assay Kit (Bio-Rad, Hercules, CA). Protein (10 g) was electrophoresed through a 12% denaturing polyacryamide slab gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc.) by electroblotting. Cytochrome c was detected by Western blotting using mouse monoclonal anti-cytochrome C antibody (7H8.2C12) (PharMingen) and the Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products) according to the manufacturer’s instructions. RNA isolation and Northern blot analysis. Total cellular RNA was purified using the Trizol method (GIBCO/BRL) according to the manufacturer’s instructions. For Northern blot analysis, 20 g of total RNA was separated in a 1.5% formaldehyde agarose gel and blotted to Hybond-N ⫹ membrane (Amersham Pharmacia, Piscataway, NJ). The blots were hybridized with random primed radiolabeled probes as follows: a 1.2 kb cDNA probe for DR5 (13); a 1.2 kb cDNA for TRUNDD (21); a 780 bp cDNA probe for TRID (15); and a 599 bp probe for the DR4 cytoplasmic domain (59). Radioactive signals were analyzed by autoradiography. The membrane was stripped and reprobed with a GAPDH cDNA as a loading control.
RESULTS TRAIL-induced apoptosis involves caspase activation in human non-small cell lung cancer cell lines. It has been shown that TRAIL can selectively kill transformed and tumor cells but not normal cells. To examine the effects of TRAIL on human non-small cell lung cancer cells (NSCLC), ten NSCLC cells lines were treated with recombinant TRAIL at various doses for 6 h, followed by cell survival assessment using the sulforhodamine B (SRB) assay. Figure 1A shows that the different cell lines have different sensitivity to TRAIL. For example, following a 6-h treatment, ⬃25% of H460 cells survived, compared to ⬃95% of Calu-1 cells. Survival rates for the rest of cell lines were intermediate between them. Thus, H460 is the most sensitive cell line, while Calu-1 is the most resistant cell line tested. Furthermore, we compared TRAIL sensitivities of H460 and Jurkat cells, the latter has been known to be sensitive to a variety of stimuli-induced apoptosis (40). As shown in Figs. 1B and 1C, TRAIL induced DNA fragmentation and PARP cleavage in both cell lines. However, in H460 cells, TRAIL at 25 ng/ml, induced DNA fragmentation to a higher degree and completely cleaved PARP, indicating that H460 cells are even more sensitive than Jurkat cells to TRAIL-induced apoptosis. Therefore, we chose H460 as
a system to study the mechanism of TRAIL-induced apoptosis. It has been shown that apoptosis involves activation of caspases (7). To assess whether TRAIL-induced apoptosis of H460 cells involves activation of caspases, the TRAIL-treated H460 cell lysates were analyzed by Western blot analysis for caspases 10, 9, 8, 7, and 3, as well as Bid, a pro-apoptotic member of the Bcl2 family. Cleavage of caspase 8, the apical caspase in the death receptor pathway, was detected as early as 2 h, indicating that caspase 8 is involved in the TRAIL pathway and the activation of caspase 8 is an early event (Fig. 1D). We could not detect cleavage of caspase 10 during TRAIL-induced apoptosis (Fig. 1D), implying that caspase 10 may not be required for apoptosis induction in H460 cells. Caspase 3 and 7, two executioner caspases and substrates for caspase 8, were activated (cleaved) at 2 and 4 h, respectively (Fig. 1D). As expected, complete cleavage of PARP, a hallmark of apoptosis, was detected at 4 h following TRAIL treatment (Fig. 1D). Since Bid has been shown to be a substrate for caspase 8 (40, 41), we also examined cleavage of Bid following TRAIL treatment. As shown in Fig. 1D, cleavage of Bid was detected at 2 h and increased to a higher degree at 4 h following TRAIL treatment. Since cleavage of Bid has been shown to trigger the release of cytochrome c from the mitochondria to the cytosol (30, 40), we examined the release of cytochrome c and found that cytochrome c was dramatically increased in the cytosol in cells following a 3-h treatment by TRAIL (Fig. 1E). Consequently, cleavage of caspase 9 was detected at 4 h, 2 h later than Bid cleavage (Fig. 1D), consistent with the idea that cleavage of caspase 9 occurs downstream of Bid activation in death receptor-mediated apoptosis (30, 40). These results suggest that TRAIL-mediated apoptosis in H460 cells involves activation of the mitochondrial pathway. Thus, our results are in agreement with the previous findings that TRAIL-mediated apoptosis involves a caspase cascade (27, 28). Overexpression of Bcl2 renders H460 cells resistant to TRAIL-mediated cell death. Although Bcl2 and Bcl-X L are believed to be primarily involved in suppressing apoptosis at the mitochondria induced by stimuli, including DNA damage, they have also been shown to inhibit Fas and TNFR1-mediated apoptosis (38, 39, 42, 43). To explore the possibility that Bcl2 may regulate TRAIL-induced apoptosis, H460 cells were transfected with an empty vector, pSFFV-neo, or a hemagglutinin-tagged full length Bcl2 expression vector, pSFFV-neo-HA Bcl2, and selected with G418 for clones that stably express Bcl2 protein. It is noteworthy that the pSFFV-neo-HA Bcl2 was previously shown to behave the same as wild type Bcl2, and the addition of the HA tag to Bcl2 does not confer any selective growth advantages for cell survival (44). A Western blot analysis of Bcl2 was employed to screen for clones
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FIG. 1. H460 human NSCLC cell lines are very sensitive to TRAIL-induced apoptosis (A, B, and C) and TRAIL effectively induces activation of a caspase cascade (D) and release of cytochrome c from the mitochondria (E) in H460 cells. (A) After a 6-h treatment with the indicated concentrations of TRAIL, cell numbers were estimated by sulforhodamine B (SRB) assay. Cell survival was calculated as described in Materials and Methods. (B and C) After treatment with the indicated concentrations of TRAIL for 5 h, both floating and attached cells were harvested and counted. Five thousand cells were used for analyzing DNA fragmentation using ELISA (B) and the rest of the cells were used for preparation of whole-cell protein lysates and Western blot analysis (C) as described in Materials and Methods. (D) At the indicated times following treatment with 100 ng/ml TRAIL, both floating and attached cells were harvested and whole-cell protein lysates were prepared for Western blot analysis. Casp, caspase. (E) After treatment with 100 ng/ml TRAIL for 3 h, both floating and attached cells were harvested and cytosol fractions were prepared for detecting cytochrome c using Western blot analysis as described in Materials and Methods. Cyt. C, cytochrome c.
in which transfected Bcl2 was overexpressed. As expected, a 28 KD band, corresponding to HA Bcl2 (45), was detected in two HA Bcl2-transfected clones (Fig. 2A lanes 2 and 3 versus lane 1), but not in vectortransfected cells (lane 1). We designated these two clones H460-Bcl2-6 and H460-Bcl2-8, respectively, while the vector-transfected clone was termed H460-V. H460-Bcl2-6 expressed a higher level of Bcl2 than H460-Bcl2-8 (Fig. 2A lane 2 versus lane 3).
To test the effect of Bcl2-expression on TRAILinduced cytotoxicity, we treated H460-Bcl2-6, H460Bcl2-8, and H460-V cells with different concentrations of TRAIL and then determined cell death by evaluating morphological change, cell survival and DNA fragmentation. As shown in Fig. 3, H460-V cells undergo rapid apoptosis as evidenced by cells rounding up and detaching from the plate, an effect identical to that observed in parental H460 cells (data not shown). In
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FIG. 2. Stable overexpression of Bcl2 in H460 cells and expression of TRAIL receptors in H460-Bcl2 overexpressing clones. (A) Protein analysis of transfected H460 cells. H460 cells were transfected with either vector alone (lane 1) or Bcl2 expressing vector (lanes 2–3), followed by selection with G418 (500 g/ml) for 2 weeks. Individual clones were isolated and their cell lysates were analyzed on a 15% SDS-PAGE, transferred to nitrocellulose, and probed with a monoclonal antibody to Bcl2 (Oncogene Sciences). Clones 6 and 8 expressed a transfected Bcl2 protein (lanes 2 and 3 versus lane 1). Because there was a tag at the C-terminal of Bcl2, the size of this protein is about 28 kDa. (B) Northern blot analysis of total cellular RNA (20 g) from H460-V and H460-Bcl2-6 and -8 clones for expression of DR4, DR5, TRID, and TRUNDD (as indicated). In the bottom panel, expression of GAPDH was provided to document equivalent loading.
contrast, both H460-Bcl2-6 and -8 were resistant to TRAIL-induced cytoxicity, showing little changed in cell morphology (Fig. 3). With 50 ng/ml of TRAIL treatment, cell survival decreased to about 30% in H460-V cells as determined by the SRB assay, whereas more than 85% and 80%, respectively, of H460-Bcl2-6 and -8 cells survived (Fig. 4A). Consistent with the SRB results, DNA fragmentation, an indicator of apoptosis, correlated with cell viability, showing that little DNA fragmentation in H460-Bcl2-6 and H460-Bcl2-8 cells was observed following TRAIL treatment, whereas a significant increase in DNA fragmentation was detected in H460-V cells (Fig. 4B). It should be pointed out that although both Bcl2 expressing cell lines were resistant to TRAIL-induced apoptosis, the H460-Bcl2-6 cell line was even more resistant to TRAIL cytotoxicity than H460-Bcl2-8 (Fig. 4), which correlated with the amount of exogenous Bcl2. These results suggest that the levels of the Bcl2 protein may be important for determining a cell’s resistance to TRAIL cytotoxicity.
Our results show that overexpression of Bcl2 increased TRAIL resistance phenotype in H460 cells. It is possible that overexpression of Bcl2 and the consequent resistance to TRAIL-induced apoptosis were the mere result of G418 selection and not a direct effect of Bcl2 expression. To exclude this possibility, we analyzed the expression of TRAIL receptors in the vector and Bcl2 overexpressing cells. Our hypothesis was that if the expression of TRAIL receptors changed during the G418 selection, then there may be different response to TRAIL in H460-V, H460-Bcl2-6, and -8, thus conferring resistance to TRAIL-induced apoptosis. Figure 2B shows that the expression of the four TRAIL receptors, DR4, DR5, TRID, and TRUNDD, remains unchanged between H460-vector and two Bcl2-transfected cells, H460-Bcl2-6 and -8. Therefore, this result suggests that the different response to TRAIL is unlikely due to different expressed levels of TRAIL receptors on H460-V, H460-Bcl2-6, and -8 cells. Furthermore, we examined the TRAIL signaling pathway in these three cell lines because it is possible that, although TRAIL receptors remain unchanged, alterations may occur downstream of the receptors, thereby resulting in the TRAIL-resistant phenotype of the H460-Bcl2-6 and -8 cells. H460-Bcl2-6 and -8 cells were treated with a high dose of TRAIL (1 mg/ml) and then
FIG. 3. Morphological changes in H460 cells transfected with either vector or Bcl-2 following TRAIL treatment. Photographs were taken after a 4 h (A) or 24 h (B) treatment with 100 ng/ml TRAIL using a Nikon microscope. Original magnification ⫻100.
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FIG. 4. Overexpression of Bcl-2 in H460 cells prevents TRAILinduced apoptosis. After a 24-h treatment with the indicated concentrations of TRAIL, cell numbers (A) and DNA fragmentation (B) were estimated by sulforhodamine B (SRB) assay (A) and the ELISA method (B), respectively.
assessed for cell survival. We found that at this dose, both Bcl2 expressing lines undergo apoptosis, suggesting that the TRAIL-mediated apoptosis pathway is intact in H460-Bcl2-6 and -8 cells (data not shown). Thus, our results indicate that the resistance of H460Bcl2-6 and -8 cells to TRAIL-induced apoptosis is a result of overexpression of Bcl2. Bcl2 regulates TRAILinduced activation of caspases. In parental H460 cells, we found that TRAIL-induced apoptosis involves activation of caspase caspases (Fig. 1D). Since H460-Bcl2-6 and -8 cells were resistant to TRAIL-induced apoptosis, we speculated that Bcl2 inhibits TRAIL-induced apoptosis through blockage of caspase activation. To this end, H460-V, H460-Bcl2-6, and -8 cells were treated with 100 ng/ml TRAIL for different times and then protein lysates were extracted for analyzing caspase activation. Consistent with the results obtained from the parental cells, Western analysis revealed that
caspases 8, 3, and 7 were cleaved in H460-V cells following TRAIL treatment as evidenced by either the disappearance of pro-caspases or the appearance of cleaved caspase subunits (Fig. 5) and subsequent cleavage of PARP (Fig. 5D). In contrast, caspase 3 was partially cleaved in both H460-Bcl2-6 and -8 cells following TRAIL treatment compared to H460-V cells (Fig. 5D). Unexpectedly, cleavage of caspase 8 was also partially blocked in H460-Bcl2-6 and -8 cells, compared to H460-V cells in which caspase 8 was completely cleaved (Fig. 5A), as observed in the parental H460 cells (Fig. 1D). Interestingly, the cleavage of caspase 7 was completely blocked in both H460-Bcl2-6 and -8 cells, compared to H460-vector cells in which the majority of caspase 7 was cleaved (Fig. 5D). Consistent with caspase 7 activation and cell viability, cleavage of PARP was also completely blocked in H460-Bcl2-6, although a partial cleavage of PARP was observed in H460-Bcl2-8 cells. In contrast, PARP was completely cleaved in H460-V cells (Fig. 5D). Thus, our results imply that caspase 7 activation and subsequent PARP cleavage may be a determinant of cell survival in H460 cells. In addition, we treated H460-Bcl2-6 and -8 cells with the synthetic retinoid CD437 (46) and found that overexpression of Bcl2 could not block CD437mediated cell death (data not shown), consistent with previous study showing that Bcl2 was unable to block CD437-induced apoptosis in human cancer cells (47). Since we observed an inhibitory effect of Bcl2 on TRAIL-induced apoptosis, we tested for Bid cleavage, cytochrome C release from mitochondria, and caspase 9 activation in H460-Bcl2 expressing cells following TRAIL treatment. We found that Bid cleavage was partially blocked and cytochrome c release was almost completely blocked in H460-Bcl2 cells, in comparison with those changes in H460-V cells (Fig. 5). As expected, caspase 9 was activated in H460-V cells following a 4 h-treatment with TRAIL, compared to H460Bcl2-6 and -8 cells in which the activation of caspase 9 was not detected (Fig. 5C). Interestingly, activation of caspase 9 was not completely blocked in H460-Bcl2-6 and -8 cells following a 12-h treatment with TRAIL (Fig. 5B). We also noted that although cleavage of caspase 9 in H460-Bcl2-6 and -8 cells was not completely blocked, there was less cleaved caspase 9 in H460-Bcl2-6 than in H460-Bcl2-8 (Fig. 5B). Again, the extent of blockage of activated caspase 9 correlated with cell survival. Thus, these results indicate that overexpression of Bcl2 can sufficiently inhibit TRAILmediated cytochrome c release and subsequently block apoptosis, probably through interfering with the cytochrome c/caspase 9/caspase 7/PARP pathway. DISCUSSION In this study, we demonstrated that TRAIL could cause apoptosis in H460 human NSCLC cells involving
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FIG. 5. Effects of TRAIL on activation of caspase cascades (A, B, C, and D) and release of cytochrome c (E) in H460 cells transfected with either vector or Bcl-2. (A–D), After treatment with 100 ng/ml TRAIL for the indicated times, both floating and attached cells were harvested and whole-cell protein lysates were prepared for detecting activation of initiator caspases (Casp-8, -9, and -10) (A, B, and C) and effector caspases (Casp-3 and -7) (D) and cleavage of their substrates Bid (A) and PARP (D) using Western blot analysis. Casp, caspase. (E) After treatment with 100 ng/ml TRAIL for the indicated times, both floating and attached cells were harvested and cytosol fractions were prepared for detecting cytochrome c using Western blot analysis as described in Materials and Methods. Cyt. C, cytochrome c.
activation of a caspase cascade. This finding was not surprising, considering that TRAIL has been shown to induce apoptosis in a variety of tumor cells (10, 11). Although TRAIL can induce apoptosis in a variety of cells, the mechanism of this activity remains unclear. It has been reported that FADD knockout cells undergo TRAIL-mediated apoptosis, and a dominant negative mutant of FADD has little effect on TRAILmediated apoptosis (22, 48). However, accumulating evidence suggests that TRAIL-mediated apoptosis utilizes FADD as the adaptor and recruits caspase 8, but not caspase 10, as the initiator caspase to the DISC,
and then transduces the apoptotic signal to downstream (27, 49). Although we did not study the requirement of adaptor in the TRAIL apoptotic pathway, we assume that FADD is recruited to the TRAIL DISC. Nevertheless, our results showed clearly that caspase 8, but not caspase 10 (Fig. 1B) was involved in TRAILmediated apoptosis. It has been shown that caspase 8 activated by death receptor ligation directly activates executioner caspases including caspases 3, 6, and 7 in Fas- and TNFR1-mediated apoptosis (50). In addition, active caspase 8 is able to cleave the pro-apoptotic Bcl2 family
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member, Bid and trigger a distinct apoptotic pathway involving the mitochondria in some cells (40, 41). Activation of the mitochondrial pathway is believed to amplify the apoptotic process induced by activation of death receptors. In this pathway, release of cytochrome c into the cytoplasm results in complex formation with Apaf1 and pro-caspase 9 leading to activation of caspase 9. As a result, the activated caspase 9 is able to activate executioner caspases. Once the executioner caspases are activated, they dismantle a variety of substrates to cause cell death accompanied by the morphological and biochemical features of apoptosis. We have shown that PARP was cleaved in TRAIL-treated H460 but not in control cells (Fig. 1D). We were also able to detect Bid cleavage, cytochrome c release, and caspase 9 activation (Fig. 1D), indicating that the mitochondrial pathway is involved in TRAIL-mediated apoptosis. Taken together, these results, along with other reports (51, 52), suggest that TRAIL induces apoptosis through activation of a caspase cascade involving mitochondrial-dependent and -independent pathways. The Bcl2 family has been shown to play an important role in regulating apoptosis. Two anti-apoptotic members, Bcl2 and Bcl-X L, have been shown to inhibit the release of cytochrome c from the mitochondria to the cytosol in response to a variety of the apoptotic stimuli (34, 35). In addition, it has been shown that overexpression of Bcl2/Bcl-XL inhibits cytochrome c release induced by the death receptors of Fas and TNFR1. In this study, we have clearly shown that Bcl2overexpressing H460 cells were resistant to TRAILinduced cell death (Figs. 3 and 4). This differs from two recently published studies in which Bcl2/Bcl-XL could not block TRAIL-induced apoptosis in Jurket cells, although both studies demonstrated Bid cleavage and cytochrome c release (51, 52). The reason for this discrepancy is not clear. We believe that this difference may be due either to differences in the expressed levels of Bcl2 protein or to intrinsic differences in the cell lines used. We believe that the levels of Bcl2 protein in the cells are important in determining TRAIL sensitivity. This is based on the following observations: 1) both H460-Bcl2-6 and -8 are resistant to TRAIL-induced apoptosis, compared to H460-V in which no exogenous Bcl2 was expressed; 2) a higher level of Bcl2 protein in H460-Bcl2-6 than in H460-Bcl2-8 cells correlated with their TRAIL sensitivity (Figs. 2A and 3); and 3) two H460 clones whose expression levels of Bcl2 protein were slightly higher than vector clones were found to have similar sensitivity to TRAIL as the vector clones (data not shown). Therefore, the difference between our results and other reports may be due to the expression levels of Bcl2. Nevertheless, we clearly showed that overexpression of Bcl2 confers H460 resistance to TRAIL-induced apoptosis. Alternatively, the discrepancy may also be explained by the different cell types used in the studies, as our study used an epithelial
solid tumor cell line, while the other studies used Jurkat and CEM cells, both are hematopoietic cells (52, 53). Generally speaking, hematopoietic cells have a greater tendency to undergo apoptosis than epithelial cells in response to apoptotic stimuli. We have shown that in Bcl2 expressing cells, especially in H460-Bcl2-6 cells, cleavage of caspase 8 was not complete. This is not surprising, given the fact that we do not fully understand the ordered activation of caspases. It is possible that executioner caspases can serve as a feedback loop by cleaving caspase 8 to amplify the apoptotic process. Because overexpression of Bcl2 partially blocks mitochondrial-mediated activation of executioner caspases (Fig. 5), activation of caspase 8 in Bcl2 expressing cells was delayed or partially blocked (Fig. 4). This result is in agreement with the report that caspase 3 could activate caspase 8 and thus amplify the apoptotic signal (53). Nevertheless, we believe that Bcl2 acts downstream of caspase 8. We found that activation of caspase 9 following a 4 h-treatment with TRAIL was not detected in H460Bcl2-6 and -8 cells (Fig. 5C), while the activation of caspase 9 was partially detected following a 12- h treatment with TRAIL (Fig. 5B), suggesting that a partial activation of caspase 9 in Bcl2-expressing cells may be indirect. Consistent with this finding, the executioner caspase 3 has been found to activate caspase 8 and 9 in the knockout system (54). Thus, the cleavage of caspase 9 observed in two Bcl2-expressing H460 cells may be due to a feedback loop through caspase 3. We have shown that activation of caspase 7, but not caspase 3, was completely blocked by overexpression of Bcl2 in H460 cells during TRAIL-mediated apoptosis (Fig. 5). The mechanism underlying this inhibition is not clear. Caspase 3 has been shown to be activated by either caspase 8 or caspase 9 (5). It has been reported that during Fas-mediated apoptosis in vivo, active caspase 7 is associated almost exclusively with the mitochondrial and microsomal fractions, whereas active caspase 3 is localized primarily to the cytosol (55). Given Bcl2/Bcl-XL localization to the cytoplasmic membranes of mitochondria, endoplasmic reticulum, and nuclei (33), it is possible that direct or indirect association of Bcl2 and caspase 7 in the same compartment of the cell results in the inactivation of caspase 7. It is not known that whether caspase 7 and Bcl2 can form a complex directly or indirectly, and this issue needs to be investigated. Consistent with our observation, activation of caspase 7 is completely blocked in TNFR1-induced apoptosis by Bcl-XL (39). In our experiments, we showed that cleavage of PARP is completely blocked in H460-Bcl2-6 cells and partially blocked in H460-Bcl2-8 cells. Although three executioner caspases have been shown to cleave specific substrates, in the case of caspase 3 and 7, they have distinct substrates but possibly overlapping roles in apoptosis, based on the in vitro study (55). It has been reported
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that PARP cleavage is primarily targeted by caspase 7, not by caspase 3 (56). Consistent with this finding, inhibition of caspase 7 activation by Bcl2 during TRAIL-mediated apoptosis impairs PARP cleavage, as we observed in this study. In summary, we have found that TRAIL effectively kills human lung cancer cells through induction of apoptosis involving a caspase cascade. Overexpression of Bcl2 blocks TRAIL-mediated caspase activation and apoptosis. Our results indicate that TRAIL may be ineffective in tumors with higher levels of Bcl2 protein, when applied as an anti-cancer agent. Future experiments will focus on analyzing the expression level of Bcl2 and TRAIL sensitivity as well as the mechanism by which Bcl2 blocks TRAIL-induced apoptosis. ACKNOWLEDGMENTS We are grateful to Dr. Lawrence Boise (University of Miami) for the HA-Bcl2 expressing vector. We also thank Dr. James Eliason for helpful discussion. This work was supported by a start-up fund from the Karmanos Cancer Institute.
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Overexpression of Bcl2 Blocks TNF-Related ApoptosisInducing Ligand (TRAIL)-Induced Apoptosis in Human Lung Cancer Cells Shi-Yong Sun,* Ping Yue,* Jun-Ying Zhou,† Yinghong Wang,† Hyeong-Reh Choi Kim,† Reuben Lotan,* and Gen Sheng Wu† ,1 *Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030; and †Program in Developmental Therapeutics, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201
Received December 20, 2000
The tumor necrosis factor (TNF) related apoptosisinducing ligand (TRAIL or Apo2L) and its receptors are members of the tumor necrosis factor superfamily. TRAIL triggers apoptosis by binding to its two proapoptotic receptors DR4 and DR5, a process which is negatively regulated by binding of TRAIL to its two decoy receptors TRID and TRUNDD. Here, we show that TRAIL effectively induces apoptosis in H460 human non-small-cell lung carcinoma cells via cleavage of caspases 8, 9, 7, 3, and BID, release of cytochrome c from the mitochondria, and cleavage of poly (ADPribose) polymerase (PARP). However, overexpression of Bcl2 blocked TRAIL-induced apoptosis in H460 cells, which correlated with the Bcl2 protein levels. Importantly, the release of cytochrome c and cleavage of caspase 7 triggered by TRAIL were considerably blocked in Bcl2 overexpressing cells as compared to vector control cells. Moreover, inhibition of TRAILmediated cytochrome c release and caspase 7 activation by Bcl2 correlated with the inability of PARP to be cleaved and the inability of the Bcl2 transfectants to undergo apoptosis. Thus, these results suggest that Bcl2 can serve an anti-apoptotic function during TRAIL-dependent apoptosis by inhibiting the release of cytochrome c and activation of caspase 7, thereby blocking caspase 7-dependent cleavage of cellular substrates. © 2001 Academic Press Key Words: TRAIL; Bcl2; caspases; apoptosis; mitochondria.
Programmed cell death or apoptosis is a genetically controlled mechanism essential for the maintenance of tissue homeostasis, proper development and the elimination of unwanted cells. In mammalian cells, two 1
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major apoptosis pathways are proposed: the first one involves signals transduced through death receptors; the second relies on a signal from the mitochondria (1, 2). Both pathways are involved in an ordered activation of a set of cysteine proteases called caspases (3), which in turn cleave cellular substrates and result in the morphological and biochemical changes characteristic of apoptosis (4, 5). In the death receptor pathway, apoptosis induction involves the engagement of a set of ligands and their corresponding receptors and then transmission of the apoptotic signal in the cytoplasm by a number of caspases (6, 7). There are a number of ligand-receptor systems and target caspases involved in apoptosis. These death-inducing receptors belong to the tumor necrosis factor (TNF) superfamily including Fas/APO1, TNFR, and TRAIL receptors, all of which are characterized by a cysteine-rich extracellular domain and an intracellular “death domain” (7). When a ligand binds to the receptor, the receptor becomes trimerized and activated. The activated receptor recruits a death domain containing adaptor and a pro-caspase 8 molecule through protein-protein interactions to form a deathinducing signaling complex (DISC) (8). FADD is directly recruited into the DISC during Fas-mediated apoptosis, while TNF-induced apoptosis involves both FADD and TRADD (7). Recruitment of pro-caspase 8 to the DISC activates caspase 8 by oligomerizationinduced autocatalytic processing (9), which results in activation of downstream caspases, such as caspases 3, 6, and 7, and induction of apoptosis (7). TRAIL, a recently identified member of the TNF family, has been shown to induce apoptosis in a variety of transformed or tumor cells but not normal cells (10, 11). To date, five human TRAIL receptors have been identified; they are DR4 (12), DR5 (KILLER, TRAIL-R1, TRICK2) (13–17), TRID (TRAIL-R3, DcR1, or LIT) (15, 18 –20), TRUNDD
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(DcR2 or TRAIL-R4) (21–23), and osteoprotegerin (24). Additionally, a mouse TRAIL receptor has been identified (25). Both human DR4 and DR5 are proapoptotic receptors and binding of TRAIL to these receptors leads to the recruitment of the adapter and induction of apoptosis through activation of caspases (7). However, unlike DR4 and DR5, TRID lacks an intracellular domain and TRUNDD has a truncated death domain. Thus, these two receptors act as decoy receptors to antagonize TRAIL-induced apoptosis by competing for ligand binding (7). The mechanism of TRAIL receptormediated apoptosis is not completely understood. A study from FADD knockout cells showed that FADD is not required for TRAIL-mediated apoptosis (26). However, recent reports suggested that TRAIL-mediated apoptosis involves activation of FADD and caspase 8 (27–29). In contrast to the death receptor pathway, apoptosis can also be triggered by the mitochondrial pathway. In response to apoptotic stimuli, such as DNA damage (including anticancer drug treatment and growth factor withdrawal), damaged mitochondria release cytochrome c into the cytosol where cytochrome c can associate with Apaf1 and procaspase 9 in the presence of dATP, leading to activation of caspase 9 (30). Subsequently, the activated caspase 9 cleaves downstream caspases, such as caspases 3, 6, and 7, leading to cell death (31). In addition, activation of caspase 8 through engagement of death receptors can also trigger the mitochondrial pathway via Bid, a pro-apoptotic member of the Bcl2 family. This activation of the mitochondrial pathway is believed to amplify death receptorinduced apoptosis. Because mitochondrial damage and subsequent cytochrome c release are critical in apoptosis induction in response to some death stimuli, studies have been carried out to elucidate the role of members of the Bcl2 family in regulating mitochondrial integrity. The Bcl2 family contains anti-apoptotic members, such as Bcl2 and Bcl-X L, and pro-apoptotic members, such as Bax and Bid. It is believed that the ratios of proapoptotic and anti-proapoptotic members determine whether cells live or die (32). Both Bcl2 and Bcl-X L are localized on the cytoplasmic side of the mitochondrial membrane, the endoplasmic reticulum, and nuclear membrane (33). It has been shown that overexpression of Bcl2/Bcl-X L can protect cells from death induced by a variety of agents, such as UV irradiation, cytotoxic drugs, growth factor withdrawal, p53, and c-myc overexpression. In addition, Bcl2/ Bcl-X L can block the release of cytochrome c and thus inhibit apoptosis (34 –36). Lastly, Bcl2 can protect some cell types from Fas and TNFR1-mediated apoptosis (37–39). In this study, we investigated the role of Bcl2 in regulating TRAIL-mediated apoptosis. Here we report that TRAIL can induce apoptosis involving a caspase
cascade including activation of caspases 8, 9, 3, 7, cleavage of Bid, release of cytochrome c from mitochondria, and cleavage of PARP in H460 cells. More importantly, we show that overexpression of Bcl2 protects H460 cells from TRAIL-induced apoptosis. Furthermore, we show that Bcl2 inhibits cytochrome c release, caspase 7 activation, and PARP cleavage induced by TRAIL. Our results suggest that if TRAIL were applied for human cancer therapy, tumors that overexpress the Bcl2 protein may not be sensitive to TRAIL-induced cell death. MATERIALS AND METHODS Cells, cell culture, and transfection conditions. Seven human NSCLC cell lines (H292, H460, H1944, H522, H1792, H596, and H157) were obtained from Dr. Adi Gazdar (University of Texas Southwestern Medical Center, Dallas, TX). Three additional lines (SK-MES-1, A549 and Calu-1) were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in a 1:1 (v/v) mixture of DMEM and Ham’s F12 medium supplemented with 5% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. To generate cell lines in which the Bcl2 gene is overexpressed, H460 cells were transfected with either pSFFV-neo, an empty vector, or pSFFVneo-HA Bcl2, a hemagglutinin-tagged full length Bcl2 expression vector (a gift from Lawrence Boise, University of Miami, Miami, FL) as previously described (44). Following 2 weeks of selection in the presence of 500 g/ml of G418 (GIBCO/BRL, Rockville, MD), individual clones were isolated as described previously (57). Clones that expressed no exogenous Bcl2 (vector control) or different levels of exogenous Bcl2 protein determined by Western blot analysis were used for this study. Cell survival assay. Cells were seeded at densities of 1 ⫻ 10 4 cells per well in 96-well tissue culture plates. Cells were treated with different concentrations of TRAIL. After treatment for the indicated times, cell numbers were estimated by the sulforhodamine B (SRB) assay as described previously (58). Cell survival was calculated by using the equation: % cell survival ⫽ (A t/A c) ⫻ 100, where A t and A c represent the absorbance in treated and control cultures, respectively. DNA fragmentation assay. Cells were plated at densities of 5 ⫻ 10 3 cells per well in 96-well cell culture plates 1 day before treatment. After a 24 h-treatment, DNA fragmentation was evaluated by examination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) using a Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions. Western blot analysis. Whole cell lysates from both attached and detached floating cells were prepared as described previously (46) and protein concentration was determined using the Protein Assay Kit (Bio-Rad, Hercules, CA). Cell lysates (50 g) were electrophoresed through 7.5–12% denaturing polyacrylamide slab gels and transferred to a PROTRAN nitrocellulose transfer and immobilization membrane (Schleicher & Schuell, Inc., Keene, NH) by electroblotting. The blots were probed or reprobed with the antibodies, and then antibody binding was detected using the Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products, Boston, MA) according to the manufacturer’s protocol. Mouse anticaspase-3 (clone 19) and mouse monoclonal anti-caspase-7 (clone B94-1) antibodies were purchased from PharMingen (San Diego, CA). Rabbit polyclonal anti-caspase-9 antibody was purchased from New England BioLabs, Inc. (Beverly, MA). Goat polyclonal anticaspase-8 (C-20) and rabbit polyclonal anti-caspase-10 (H131) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa
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Cruz, CA). Rabbit polyclonal anti-Bid antibody was purchased from Trevigen (Gaithersburg, MD). Rabbit polyclonal anti-PARP (VIC 5) and anti-actin antibodies were purchased from Roche Molecular Biochemicals and Sigma Chemical Co. (St. Louis, MO), respectively. Measurement of cytochrome c release. Cells were plated onto 10-cm diameter dishes one day before treatment. After the cells were exposed to TRAIL for the indicated times, both floating and attached cells were harvested and washed once with PBS. The cell pellets were resuspended in buffer containing 0.25 M sucrose, 30 mM TrisHCl, pH 7.9, 1 mM EDTA and protease inhibitor cocktail (PharMingen) and homogenized with a glass dounce homogenizer and a B pestle (30 strokes). After centrifugation at 14,000 rpm for 15 min, the supernatants were transferred to another ultracentrifuge tube and centrifuged at 100,000 g for 30 min. The supernatants were then collected and stored at ⫺80°C. Protein concentration was determined with the Protein Assay Kit (Bio-Rad, Hercules, CA). Protein (10 g) was electrophoresed through a 12% denaturing polyacryamide slab gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc.) by electroblotting. Cytochrome c was detected by Western blotting using mouse monoclonal anti-cytochrome C antibody (7H8.2C12) (PharMingen) and the Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products) according to the manufacturer’s instructions. RNA isolation and Northern blot analysis. Total cellular RNA was purified using the Trizol method (GIBCO/BRL) according to the manufacturer’s instructions. For Northern blot analysis, 20 g of total RNA was separated in a 1.5% formaldehyde agarose gel and blotted to Hybond-N ⫹ membrane (Amersham Pharmacia, Piscataway, NJ). The blots were hybridized with random primed radiolabeled probes as follows: a 1.2 kb cDNA probe for DR5 (13); a 1.2 kb cDNA for TRUNDD (21); a 780 bp cDNA probe for TRID (15); and a 599 bp probe for the DR4 cytoplasmic domain (59). Radioactive signals were analyzed by autoradiography. The membrane was stripped and reprobed with a GAPDH cDNA as a loading control.
RESULTS TRAIL-induced apoptosis involves caspase activation in human non-small cell lung cancer cell lines. It has been shown that TRAIL can selectively kill transformed and tumor cells but not normal cells. To examine the effects of TRAIL on human non-small cell lung cancer cells (NSCLC), ten NSCLC cells lines were treated with recombinant TRAIL at various doses for 6 h, followed by cell survival assessment using the sulforhodamine B (SRB) assay. Figure 1A shows that the different cell lines have different sensitivity to TRAIL. For example, following a 6-h treatment, ⬃25% of H460 cells survived, compared to ⬃95% of Calu-1 cells. Survival rates for the rest of cell lines were intermediate between them. Thus, H460 is the most sensitive cell line, while Calu-1 is the most resistant cell line tested. Furthermore, we compared TRAIL sensitivities of H460 and Jurkat cells, the latter has been known to be sensitive to a variety of stimuli-induced apoptosis (40). As shown in Figs. 1B and 1C, TRAIL induced DNA fragmentation and PARP cleavage in both cell lines. However, in H460 cells, TRAIL at 25 ng/ml, induced DNA fragmentation to a higher degree and completely cleaved PARP, indicating that H460 cells are even more sensitive than Jurkat cells to TRAIL-induced apoptosis. Therefore, we chose H460 as
a system to study the mechanism of TRAIL-induced apoptosis. It has been shown that apoptosis involves activation of caspases (7). To assess whether TRAIL-induced apoptosis of H460 cells involves activation of caspases, the TRAIL-treated H460 cell lysates were analyzed by Western blot analysis for caspases 10, 9, 8, 7, and 3, as well as Bid, a pro-apoptotic member of the Bcl2 family. Cleavage of caspase 8, the apical caspase in the death receptor pathway, was detected as early as 2 h, indicating that caspase 8 is involved in the TRAIL pathway and the activation of caspase 8 is an early event (Fig. 1D). We could not detect cleavage of caspase 10 during TRAIL-induced apoptosis (Fig. 1D), implying that caspase 10 may not be required for apoptosis induction in H460 cells. Caspase 3 and 7, two executioner caspases and substrates for caspase 8, were activated (cleaved) at 2 and 4 h, respectively (Fig. 1D). As expected, complete cleavage of PARP, a hallmark of apoptosis, was detected at 4 h following TRAIL treatment (Fig. 1D). Since Bid has been shown to be a substrate for caspase 8 (40, 41), we also examined cleavage of Bid following TRAIL treatment. As shown in Fig. 1D, cleavage of Bid was detected at 2 h and increased to a higher degree at 4 h following TRAIL treatment. Since cleavage of Bid has been shown to trigger the release of cytochrome c from the mitochondria to the cytosol (30, 40), we examined the release of cytochrome c and found that cytochrome c was dramatically increased in the cytosol in cells following a 3-h treatment by TRAIL (Fig. 1E). Consequently, cleavage of caspase 9 was detected at 4 h, 2 h later than Bid cleavage (Fig. 1D), consistent with the idea that cleavage of caspase 9 occurs downstream of Bid activation in death receptor-mediated apoptosis (30, 40). These results suggest that TRAIL-mediated apoptosis in H460 cells involves activation of the mitochondrial pathway. Thus, our results are in agreement with the previous findings that TRAIL-mediated apoptosis involves a caspase cascade (27, 28). Overexpression of Bcl2 renders H460 cells resistant to TRAIL-mediated cell death. Although Bcl2 and Bcl-X L are believed to be primarily involved in suppressing apoptosis at the mitochondria induced by stimuli, including DNA damage, they have also been shown to inhibit Fas and TNFR1-mediated apoptosis (38, 39, 42, 43). To explore the possibility that Bcl2 may regulate TRAIL-induced apoptosis, H460 cells were transfected with an empty vector, pSFFV-neo, or a hemagglutinin-tagged full length Bcl2 expression vector, pSFFV-neo-HA Bcl2, and selected with G418 for clones that stably express Bcl2 protein. It is noteworthy that the pSFFV-neo-HA Bcl2 was previously shown to behave the same as wild type Bcl2, and the addition of the HA tag to Bcl2 does not confer any selective growth advantages for cell survival (44). A Western blot analysis of Bcl2 was employed to screen for clones
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FIG. 1. H460 human NSCLC cell lines are very sensitive to TRAIL-induced apoptosis (A, B, and C) and TRAIL effectively induces activation of a caspase cascade (D) and release of cytochrome c from the mitochondria (E) in H460 cells. (A) After a 6-h treatment with the indicated concentrations of TRAIL, cell numbers were estimated by sulforhodamine B (SRB) assay. Cell survival was calculated as described in Materials and Methods. (B and C) After treatment with the indicated concentrations of TRAIL for 5 h, both floating and attached cells were harvested and counted. Five thousand cells were used for analyzing DNA fragmentation using ELISA (B) and the rest of the cells were used for preparation of whole-cell protein lysates and Western blot analysis (C) as described in Materials and Methods. (D) At the indicated times following treatment with 100 ng/ml TRAIL, both floating and attached cells were harvested and whole-cell protein lysates were prepared for Western blot analysis. Casp, caspase. (E) After treatment with 100 ng/ml TRAIL for 3 h, both floating and attached cells were harvested and cytosol fractions were prepared for detecting cytochrome c using Western blot analysis as described in Materials and Methods. Cyt. C, cytochrome c.
in which transfected Bcl2 was overexpressed. As expected, a 28 KD band, corresponding to HA Bcl2 (45), was detected in two HA Bcl2-transfected clones (Fig. 2A lanes 2 and 3 versus lane 1), but not in vectortransfected cells (lane 1). We designated these two clones H460-Bcl2-6 and H460-Bcl2-8, respectively, while the vector-transfected clone was termed H460-V. H460-Bcl2-6 expressed a higher level of Bcl2 than H460-Bcl2-8 (Fig. 2A lane 2 versus lane 3).
To test the effect of Bcl2-expression on TRAILinduced cytotoxicity, we treated H460-Bcl2-6, H460Bcl2-8, and H460-V cells with different concentrations of TRAIL and then determined cell death by evaluating morphological change, cell survival and DNA fragmentation. As shown in Fig. 3, H460-V cells undergo rapid apoptosis as evidenced by cells rounding up and detaching from the plate, an effect identical to that observed in parental H460 cells (data not shown). In
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FIG. 2. Stable overexpression of Bcl2 in H460 cells and expression of TRAIL receptors in H460-Bcl2 overexpressing clones. (A) Protein analysis of transfected H460 cells. H460 cells were transfected with either vector alone (lane 1) or Bcl2 expressing vector (lanes 2–3), followed by selection with G418 (500 g/ml) for 2 weeks. Individual clones were isolated and their cell lysates were analyzed on a 15% SDS-PAGE, transferred to nitrocellulose, and probed with a monoclonal antibody to Bcl2 (Oncogene Sciences). Clones 6 and 8 expressed a transfected Bcl2 protein (lanes 2 and 3 versus lane 1). Because there was a tag at the C-terminal of Bcl2, the size of this protein is about 28 kDa. (B) Northern blot analysis of total cellular RNA (20 g) from H460-V and H460-Bcl2-6 and -8 clones for expression of DR4, DR5, TRID, and TRUNDD (as indicated). In the bottom panel, expression of GAPDH was provided to document equivalent loading.
contrast, both H460-Bcl2-6 and -8 were resistant to TRAIL-induced cytoxicity, showing little changed in cell morphology (Fig. 3). With 50 ng/ml of TRAIL treatment, cell survival decreased to about 30% in H460-V cells as determined by the SRB assay, whereas more than 85% and 80%, respectively, of H460-Bcl2-6 and -8 cells survived (Fig. 4A). Consistent with the SRB results, DNA fragmentation, an indicator of apoptosis, correlated with cell viability, showing that little DNA fragmentation in H460-Bcl2-6 and H460-Bcl2-8 cells was observed following TRAIL treatment, whereas a significant increase in DNA fragmentation was detected in H460-V cells (Fig. 4B). It should be pointed out that although both Bcl2 expressing cell lines were resistant to TRAIL-induced apoptosis, the H460-Bcl2-6 cell line was even more resistant to TRAIL cytotoxicity than H460-Bcl2-8 (Fig. 4), which correlated with the amount of exogenous Bcl2. These results suggest that the levels of the Bcl2 protein may be important for determining a cell’s resistance to TRAIL cytotoxicity.
Our results show that overexpression of Bcl2 increased TRAIL resistance phenotype in H460 cells. It is possible that overexpression of Bcl2 and the consequent resistance to TRAIL-induced apoptosis were the mere result of G418 selection and not a direct effect of Bcl2 expression. To exclude this possibility, we analyzed the expression of TRAIL receptors in the vector and Bcl2 overexpressing cells. Our hypothesis was that if the expression of TRAIL receptors changed during the G418 selection, then there may be different response to TRAIL in H460-V, H460-Bcl2-6, and -8, thus conferring resistance to TRAIL-induced apoptosis. Figure 2B shows that the expression of the four TRAIL receptors, DR4, DR5, TRID, and TRUNDD, remains unchanged between H460-vector and two Bcl2-transfected cells, H460-Bcl2-6 and -8. Therefore, this result suggests that the different response to TRAIL is unlikely due to different expressed levels of TRAIL receptors on H460-V, H460-Bcl2-6, and -8 cells. Furthermore, we examined the TRAIL signaling pathway in these three cell lines because it is possible that, although TRAIL receptors remain unchanged, alterations may occur downstream of the receptors, thereby resulting in the TRAIL-resistant phenotype of the H460-Bcl2-6 and -8 cells. H460-Bcl2-6 and -8 cells were treated with a high dose of TRAIL (1 mg/ml) and then
FIG. 3. Morphological changes in H460 cells transfected with either vector or Bcl-2 following TRAIL treatment. Photographs were taken after a 4 h (A) or 24 h (B) treatment with 100 ng/ml TRAIL using a Nikon microscope. Original magnification ⫻100.
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FIG. 4. Overexpression of Bcl-2 in H460 cells prevents TRAILinduced apoptosis. After a 24-h treatment with the indicated concentrations of TRAIL, cell numbers (A) and DNA fragmentation (B) were estimated by sulforhodamine B (SRB) assay (A) and the ELISA method (B), respectively.
assessed for cell survival. We found that at this dose, both Bcl2 expressing lines undergo apoptosis, suggesting that the TRAIL-mediated apoptosis pathway is intact in H460-Bcl2-6 and -8 cells (data not shown). Thus, our results indicate that the resistance of H460Bcl2-6 and -8 cells to TRAIL-induced apoptosis is a result of overexpression of Bcl2. Bcl2 regulates TRAILinduced activation of caspases. In parental H460 cells, we found that TRAIL-induced apoptosis involves activation of caspase caspases (Fig. 1D). Since H460-Bcl2-6 and -8 cells were resistant to TRAIL-induced apoptosis, we speculated that Bcl2 inhibits TRAIL-induced apoptosis through blockage of caspase activation. To this end, H460-V, H460-Bcl2-6, and -8 cells were treated with 100 ng/ml TRAIL for different times and then protein lysates were extracted for analyzing caspase activation. Consistent with the results obtained from the parental cells, Western analysis revealed that
caspases 8, 3, and 7 were cleaved in H460-V cells following TRAIL treatment as evidenced by either the disappearance of pro-caspases or the appearance of cleaved caspase subunits (Fig. 5) and subsequent cleavage of PARP (Fig. 5D). In contrast, caspase 3 was partially cleaved in both H460-Bcl2-6 and -8 cells following TRAIL treatment compared to H460-V cells (Fig. 5D). Unexpectedly, cleavage of caspase 8 was also partially blocked in H460-Bcl2-6 and -8 cells, compared to H460-V cells in which caspase 8 was completely cleaved (Fig. 5A), as observed in the parental H460 cells (Fig. 1D). Interestingly, the cleavage of caspase 7 was completely blocked in both H460-Bcl2-6 and -8 cells, compared to H460-vector cells in which the majority of caspase 7 was cleaved (Fig. 5D). Consistent with caspase 7 activation and cell viability, cleavage of PARP was also completely blocked in H460-Bcl2-6, although a partial cleavage of PARP was observed in H460-Bcl2-8 cells. In contrast, PARP was completely cleaved in H460-V cells (Fig. 5D). Thus, our results imply that caspase 7 activation and subsequent PARP cleavage may be a determinant of cell survival in H460 cells. In addition, we treated H460-Bcl2-6 and -8 cells with the synthetic retinoid CD437 (46) and found that overexpression of Bcl2 could not block CD437mediated cell death (data not shown), consistent with previous study showing that Bcl2 was unable to block CD437-induced apoptosis in human cancer cells (47). Since we observed an inhibitory effect of Bcl2 on TRAIL-induced apoptosis, we tested for Bid cleavage, cytochrome C release from mitochondria, and caspase 9 activation in H460-Bcl2 expressing cells following TRAIL treatment. We found that Bid cleavage was partially blocked and cytochrome c release was almost completely blocked in H460-Bcl2 cells, in comparison with those changes in H460-V cells (Fig. 5). As expected, caspase 9 was activated in H460-V cells following a 4 h-treatment with TRAIL, compared to H460Bcl2-6 and -8 cells in which the activation of caspase 9 was not detected (Fig. 5C). Interestingly, activation of caspase 9 was not completely blocked in H460-Bcl2-6 and -8 cells following a 12-h treatment with TRAIL (Fig. 5B). We also noted that although cleavage of caspase 9 in H460-Bcl2-6 and -8 cells was not completely blocked, there was less cleaved caspase 9 in H460-Bcl2-6 than in H460-Bcl2-8 (Fig. 5B). Again, the extent of blockage of activated caspase 9 correlated with cell survival. Thus, these results indicate that overexpression of Bcl2 can sufficiently inhibit TRAILmediated cytochrome c release and subsequently block apoptosis, probably through interfering with the cytochrome c/caspase 9/caspase 7/PARP pathway. DISCUSSION In this study, we demonstrated that TRAIL could cause apoptosis in H460 human NSCLC cells involving
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FIG. 5. Effects of TRAIL on activation of caspase cascades (A, B, C, and D) and release of cytochrome c (E) in H460 cells transfected with either vector or Bcl-2. (A–D), After treatment with 100 ng/ml TRAIL for the indicated times, both floating and attached cells were harvested and whole-cell protein lysates were prepared for detecting activation of initiator caspases (Casp-8, -9, and -10) (A, B, and C) and effector caspases (Casp-3 and -7) (D) and cleavage of their substrates Bid (A) and PARP (D) using Western blot analysis. Casp, caspase. (E) After treatment with 100 ng/ml TRAIL for the indicated times, both floating and attached cells were harvested and cytosol fractions were prepared for detecting cytochrome c using Western blot analysis as described in Materials and Methods. Cyt. C, cytochrome c.
activation of a caspase cascade. This finding was not surprising, considering that TRAIL has been shown to induce apoptosis in a variety of tumor cells (10, 11). Although TRAIL can induce apoptosis in a variety of cells, the mechanism of this activity remains unclear. It has been reported that FADD knockout cells undergo TRAIL-mediated apoptosis, and a dominant negative mutant of FADD has little effect on TRAILmediated apoptosis (22, 48). However, accumulating evidence suggests that TRAIL-mediated apoptosis utilizes FADD as the adaptor and recruits caspase 8, but not caspase 10, as the initiator caspase to the DISC,
and then transduces the apoptotic signal to downstream (27, 49). Although we did not study the requirement of adaptor in the TRAIL apoptotic pathway, we assume that FADD is recruited to the TRAIL DISC. Nevertheless, our results showed clearly that caspase 8, but not caspase 10 (Fig. 1B) was involved in TRAILmediated apoptosis. It has been shown that caspase 8 activated by death receptor ligation directly activates executioner caspases including caspases 3, 6, and 7 in Fas- and TNFR1-mediated apoptosis (50). In addition, active caspase 8 is able to cleave the pro-apoptotic Bcl2 family
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member, Bid and trigger a distinct apoptotic pathway involving the mitochondria in some cells (40, 41). Activation of the mitochondrial pathway is believed to amplify the apoptotic process induced by activation of death receptors. In this pathway, release of cytochrome c into the cytoplasm results in complex formation with Apaf1 and pro-caspase 9 leading to activation of caspase 9. As a result, the activated caspase 9 is able to activate executioner caspases. Once the executioner caspases are activated, they dismantle a variety of substrates to cause cell death accompanied by the morphological and biochemical features of apoptosis. We have shown that PARP was cleaved in TRAIL-treated H460 but not in control cells (Fig. 1D). We were also able to detect Bid cleavage, cytochrome c release, and caspase 9 activation (Fig. 1D), indicating that the mitochondrial pathway is involved in TRAIL-mediated apoptosis. Taken together, these results, along with other reports (51, 52), suggest that TRAIL induces apoptosis through activation of a caspase cascade involving mitochondrial-dependent and -independent pathways. The Bcl2 family has been shown to play an important role in regulating apoptosis. Two anti-apoptotic members, Bcl2 and Bcl-X L, have been shown to inhibit the release of cytochrome c from the mitochondria to the cytosol in response to a variety of the apoptotic stimuli (34, 35). In addition, it has been shown that overexpression of Bcl2/Bcl-XL inhibits cytochrome c release induced by the death receptors of Fas and TNFR1. In this study, we have clearly shown that Bcl2overexpressing H460 cells were resistant to TRAILinduced cell death (Figs. 3 and 4). This differs from two recently published studies in which Bcl2/Bcl-XL could not block TRAIL-induced apoptosis in Jurket cells, although both studies demonstrated Bid cleavage and cytochrome c release (51, 52). The reason for this discrepancy is not clear. We believe that this difference may be due either to differences in the expressed levels of Bcl2 protein or to intrinsic differences in the cell lines used. We believe that the levels of Bcl2 protein in the cells are important in determining TRAIL sensitivity. This is based on the following observations: 1) both H460-Bcl2-6 and -8 are resistant to TRAIL-induced apoptosis, compared to H460-V in which no exogenous Bcl2 was expressed; 2) a higher level of Bcl2 protein in H460-Bcl2-6 than in H460-Bcl2-8 cells correlated with their TRAIL sensitivity (Figs. 2A and 3); and 3) two H460 clones whose expression levels of Bcl2 protein were slightly higher than vector clones were found to have similar sensitivity to TRAIL as the vector clones (data not shown). Therefore, the difference between our results and other reports may be due to the expression levels of Bcl2. Nevertheless, we clearly showed that overexpression of Bcl2 confers H460 resistance to TRAIL-induced apoptosis. Alternatively, the discrepancy may also be explained by the different cell types used in the studies, as our study used an epithelial
solid tumor cell line, while the other studies used Jurkat and CEM cells, both are hematopoietic cells (52, 53). Generally speaking, hematopoietic cells have a greater tendency to undergo apoptosis than epithelial cells in response to apoptotic stimuli. We have shown that in Bcl2 expressing cells, especially in H460-Bcl2-6 cells, cleavage of caspase 8 was not complete. This is not surprising, given the fact that we do not fully understand the ordered activation of caspases. It is possible that executioner caspases can serve as a feedback loop by cleaving caspase 8 to amplify the apoptotic process. Because overexpression of Bcl2 partially blocks mitochondrial-mediated activation of executioner caspases (Fig. 5), activation of caspase 8 in Bcl2 expressing cells was delayed or partially blocked (Fig. 4). This result is in agreement with the report that caspase 3 could activate caspase 8 and thus amplify the apoptotic signal (53). Nevertheless, we believe that Bcl2 acts downstream of caspase 8. We found that activation of caspase 9 following a 4 h-treatment with TRAIL was not detected in H460Bcl2-6 and -8 cells (Fig. 5C), while the activation of caspase 9 was partially detected following a 12- h treatment with TRAIL (Fig. 5B), suggesting that a partial activation of caspase 9 in Bcl2-expressing cells may be indirect. Consistent with this finding, the executioner caspase 3 has been found to activate caspase 8 and 9 in the knockout system (54). Thus, the cleavage of caspase 9 observed in two Bcl2-expressing H460 cells may be due to a feedback loop through caspase 3. We have shown that activation of caspase 7, but not caspase 3, was completely blocked by overexpression of Bcl2 in H460 cells during TRAIL-mediated apoptosis (Fig. 5). The mechanism underlying this inhibition is not clear. Caspase 3 has been shown to be activated by either caspase 8 or caspase 9 (5). It has been reported that during Fas-mediated apoptosis in vivo, active caspase 7 is associated almost exclusively with the mitochondrial and microsomal fractions, whereas active caspase 3 is localized primarily to the cytosol (55). Given Bcl2/Bcl-XL localization to the cytoplasmic membranes of mitochondria, endoplasmic reticulum, and nuclei (33), it is possible that direct or indirect association of Bcl2 and caspase 7 in the same compartment of the cell results in the inactivation of caspase 7. It is not known that whether caspase 7 and Bcl2 can form a complex directly or indirectly, and this issue needs to be investigated. Consistent with our observation, activation of caspase 7 is completely blocked in TNFR1-induced apoptosis by Bcl-XL (39). In our experiments, we showed that cleavage of PARP is completely blocked in H460-Bcl2-6 cells and partially blocked in H460-Bcl2-8 cells. Although three executioner caspases have been shown to cleave specific substrates, in the case of caspase 3 and 7, they have distinct substrates but possibly overlapping roles in apoptosis, based on the in vitro study (55). It has been reported
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that PARP cleavage is primarily targeted by caspase 7, not by caspase 3 (56). Consistent with this finding, inhibition of caspase 7 activation by Bcl2 during TRAIL-mediated apoptosis impairs PARP cleavage, as we observed in this study. In summary, we have found that TRAIL effectively kills human lung cancer cells through induction of apoptosis involving a caspase cascade. Overexpression of Bcl2 blocks TRAIL-mediated caspase activation and apoptosis. Our results indicate that TRAIL may be ineffective in tumors with higher levels of Bcl2 protein, when applied as an anti-cancer agent. Future experiments will focus on analyzing the expression level of Bcl2 and TRAIL sensitivity as well as the mechanism by which Bcl2 blocks TRAIL-induced apoptosis. ACKNOWLEDGMENTS We are grateful to Dr. Lawrence Boise (University of Miami) for the HA-Bcl2 expressing vector. We also thank Dr. James Eliason for helpful discussion. This work was supported by a start-up fund from the Karmanos Cancer Institute.
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