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The Caspase 9 Inhibitor Z-LEHD-FMK Protects Human Liver Cells while Permitting Death of Cancer Cells Exposed to Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Laboratory of Molecular Oncology and Cell Cycle Regulation [N. Ö., K. K., T. F. B., D. T. D., W. S. E-D.], Howard Hughes Medical Institute [D. T. D., W. S. E-D.], Departments of Medicine [K. K., W. S. E-D.], Molecular and Cellular Engineering [A. D. M.], Genetics [W. S. E-D.], Pharmacology [W. S. E-D.], Cancer Center [W. S. E-D.], and Institute for Human Gene Therapy [A. D. M., W. S. E-D.], University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

	ABSTRACT

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis of transformed and cancer cells but not of most normal cells. Recent studies have revealed an unforeseen toxicity of TRAIL toward normal human hepatocytes, thereby bringing into question the safety of systemic administration of TRAIL in humans with cancer. We found that SW480 colon adenocarcinoma, or H460 non-small cell lung cancer cell lines, which are sensitive to TRAIL, were not protected by the caspase 9 inhibitor Z-LEHD-FMK from TRAIL-induced apoptosis. However, a human colon cancer cell line HCT116 and a human embryonic kidney cell line 293, which are sensitive to TRAIL, were protected by Z-LEHD-FMK from TRAIL-mediated death. Both HCT116 and SW480 cells were protected from TRAIL by the caspase 8 inhibitor Z-IETD-FMK, dominant-negative FADD and cellular FLIP-s and interestingly both cell lines displayed caspase 9 cleavage to a similar extent after TRAIL exposure. We confirmed that normal human liver cells are sensitive to TRAIL. Moreover, we found that normal human liver cells could be protected from TRAIL-induced apoptosis by simultaneous exposure to Z-LEHD-FMK. A similar brief exposure to TRAIL plus Z-LEHD-FMK inhibited colony growth of SW480 but not HCT116 cells. Because some cancer cell lines are not protected from TRAIL-mediated killing by Z-LEHD-FMK, we believe that a brief period of caspase 9 inhibition during TRAIL administration may widen the therapeutic window and allow cancer cell killing while protecting normal liver cells. This strategy could be further developed in the effort to advance TRAIL into clinical trials.

	Introduction

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The TNF³ -related apoptosis-inducing ligand TRAIL or APO2L is a promising agent for development as a cancer therapeutic (1) . TRAIL appears to specifically kill transformed and cancer cells, whereas most normal cells appear to be resistant to TRAIL (2 , 3) . TRAIL induces apoptotic death on binding to either of two proapoptotic TRAIL receptors, TRAIL R1 (DR4 or TNFR1SF10A) or TRAIL R2 (KILLER/DR5 or TNFR1SF10B). Normal cells are believed to be resistant to TRAIL because of expressing higher levels of TRAIL decoy receptors TRID (DcR1) or TRUNDD (DcR2) on their cell surface (1 , 2) . Studies have shown that systemic administration of TRAIL is safe in mice and can kill breast or colon xenografted tumors and prolong survival (3) . In addition, it has been shown that the combination of TRAIL and chemotherapeutic agents or radiation (4) can lead to enhanced cell killing, in part, through up-regulation of KILLER/DR5 expression in wild-type p53-expressing cells (5 , 6) . However, not all cancer cells are sensitive to TRAIL (5 , 7 , 8) . A number of TRAIL-resistance mechanisms have been described in cancer cells including low or undetectable expression of DR4, high expression of FLIP, or loss of caspase 8 expression (5 , 7 , 9 , 10) .

The potential utility and safety of systemic administration of TRAIL has recently been questioned because of results showing sensitivity of human but not monkey or mouse hepatocytes to recombinant human TRAIL in vitro (11 , 12) . In the present studies, we investigated the relative contribution of caspase 8 versus caspase 9 activity toward TRAIL-mediated cytotoxicity using caspase inhibitors. We found that the killing of some but not all human cancer cell lines exposed to TRAIL could be efficiently inhibited by the caspase 9 inhibitor, Z-LEHD-FMK. This presented an important clue that some cells may be more dependent on the mitochondrial pathway and utilization of caspase 9 to achieve the execution phase of cell death. In this regard, there is evidence in the case of Fas ligand, that some cells die by a mechanism that relies on caspase 9 activation, downstream from an inefficient caspase 8 activation (13 , 14) . This so-called "type II" mechanism involves a pathway wherein caspase 8 activation leads to cytoplasmic Bid cleavage and mitochondrial translocation leading to cytochrome c release and caspase 9 activation, which is inhibitable by Bcl2. Moreover, hepatocytes from Bid-deficient mice were reported to be resistant to the cytotoxic effects of Fas (15) , supplying in vivo evidence for the hypothesis that some organs in the mammalian body may depend more heavily on the mitochondria for the execution of apoptosis initiated by death receptors (13) . It has been shown that Bid is cleaved in TRAIL-treated cells (16) . However, a recent study showed that Bcl2 or Bcl-X_L expressing cells may not be resistant to killing by TRAIL (17) . Nevertheless, we tested the effects of the caspase 9 inhibitor, Z-LEHD-FMK, on the killing of human hepatocytes exposed to TRAIL. Our results reveal that human hepatocytes are sensitive to killing by TRAIL and that coexposure to a caspase 9 inhibitor offers protection from TRAIL-mediated apoptosis. Further development of this strategy may be of value in the clinical development of TRAIL as a therapeutic agent.

	Materials and Methods

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Cancer Cell Lines.
The human colon cancer cell line SW480, and 293 human embryonic kidney cells were obtained from the American Type Culture Corporation (Manassas, VA). The human colon cancer cell line HCT116 was obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD), and the human non-small cell lung cancer cell line H460 was obtained from Dr. Stephen Baylin (Johns Hopkins University). The cells were cultured as described previously (18) .

Normal Human Hepatocyte Culture.
Normal human hepatocytes obtained from a donor liver found unsuitable for transplantation were cultured as described previously (19 , 20) with only minor modifications. The donor liver, supplied by the National Disease Research Interchange (NDRI, Philadelphia, PA), was obtained from a 76-year-old female, who died of a myocardial infarction. Briefly, the right lateral lobe (300 g) of the human liver was perfused, after cannulation of the portal vein branch, with 500 ml of PBS, complemented with antibiotics and antimycotics, at a rate of 80 ml/min, to flush out debris and RBCs. All solutions used for perfusion were warmed up to 37°C by passing the silicone tubing through a heated water bath, saturated with O₂. The lobe was then perfused with 500 ml of 2 mM EDTA solution (pH 7.4) for 6 min, followed by perfusion/recirculation with 200 ml of 0.25 mg/ml Collagenase P (pH 7.5; Boehringer Mannheim, Mannheim, Germany) digestion media. The same media was recirculated through the lobe at a rate of 80 ml/min, until a total amount of 800 ml of digestion solution was perfused. When the liver fragment appeared grossly disrupted, freed hepatocytes were suspended in DMEM and filtered through a 100 µm pore size nylon mesh (Spectrum Laboratory Products, Los Angeles, CA) and washed four times by sedimentation with ice-cold (4°C) DMEM (at 90 x g, 3 min each time). Viable hepatocytes were then purified by Percoll isodensity sedimentation. Briefly, the collected hepatocyte suspension (5 x 10⁶ cells/ml in DMEM) was diluted 1:1 with a Percoll solution [90% v/v Percoll, supplemented with salts and phosphate (pH 7.5), Pharmacia], centrifuged at 90 x g for 10 min; the pelleted, viable hepatocytes were washed three times with DMEM. The cells were counted and examined for trypan blue exclusion; cells were >95% viable. A total of 1 x 10⁶ cells/ml resuspended in attachment media (DMEM/10% FBS) were placed in primary culture using 60 mm of Vitrogen-100 collagen (CELTRIX, Santa Clara, CA)-coated Permanox plates (Nunc). After 4 h, the cells attached, and the media was changed to hormonally defined HD-DMEM, without FBS. The cells were cultured in a 5% CO₂, 37°C incubator and were used within 17 h for the experiment in Fig. 3 .

View larger version (98K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The caspase 9 inhibitor Z-LEHD-FMK protects normal human hepatocytes from TRAIL-induced apoptosis. Normal human hepatocytes were treated with TRAIL (50 ng/ml) and/or the caspase 9 inhibitor for 6 h. Annexin V-EGFP staining was performed on the cells in situ. B, quantification of the results in A.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Differential effects of caspase 8 (Z-IETD-FMK) and caspase 9 (Z-LEHD-FMK) inhibitors on TRAIL-induced apoptosis. Apoptosis occurs through FADD and procaspase 8. A, cells (as indicated) were pretreated for 30 min with the caspase 8 (C8I) and caspase 9 (C9I) inhibitors (20 µM), after which TRAIL (20ng/ml) was added. The results were analyzed after 4 h, and apoptosis was assessed using the Annexin V-EGFP assay. The experiments were carried out in triplicates (n = 3) and the SE is given for each condition. B, HCT116 and SW480 cells (as indicated) were pretreated with the inhibitors Z-IETD-FMK (caspase 8 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), Z-VDAVD-FMK (caspase 2 inhibitor), and Z-AEVD-FMK (caspase 10 inhibitor; 20 µM each) for 2 h, after which TRAIL (50ng/ml) was added. After 4 h (or 16 h for caspase 3 activation) of TRAIL exposure, extracts were collected and analyzed by Western blotting for the expression of the different proteins as indicated. C, cells were cotransfected with vector, DN-FADD, and cFLIP-s in the presence of pEGFPN1-Spectrin; treated with TRAIL (20 ng/ml) for 4 h, and the number of cells with active caspase 3 was determined by flow cytometry. The rabbit anti-active caspase 3 mAb was used to assay for active caspase 3. Phycoerythrin-conjugated goat-antirabbit mAb was used to fluorescently label the primary Ab. Experiments were performed in triplicates; bars, SE.

Annexin V-EGFP Assays by Flow Cytometry and Fluorescence Microscopy.
Cells treated with TRAIL in the presence or absence of caspase inhibitors, as well as control untreated cells, were stained with Annexin V-EGFP for analysis of phosphoserine inversion. The Annexin V-EGFP Apoptosis Detection Kit was obtained from BioVision Research Products (Palo Alto, CA) and was used as recommended by the manufacturer.

Flow cytometry was carried out using a Coulter Epics Elite Flow Cytometer. For the experiment with normal human liver cells, at the end of the treatment, 20 µl of Annexin V-EGFP solution was added directly onto the cells, in the presence of normal growth medium and the different agents, in a total volume of 2 ml. Fluorescence and the corresponding bright field images were saved, and the number of Annexin V-EGFP-positive and the total number of cells in each field was determined by counting the cells directly.

Detection of Cleaved Caspase 3 in Apoptotic Cells by Flow Cytometry.
For the experiments using cotransfected DN-FADD or cFLIP-s along with pEGFPN1-Spectrin, caspase activation was measured as a marker of apoptosis. For this assay, we used 0.125 µg/ml rabbit anti-active caspase 3 monoclonal Ab (clone C92–605; PharMingen, San Diego, CA) and the Cytofix/Cytoperm kit (PharMingen). To detect active caspase 3 by flow cytometry, 0.125 µg/ml phycoerythrin-conjugated goat antirabbit secondary Ab (Caltag Laboratories, Burlingame, CA) was used.

Colony Assays.
A total of 5000 HCT116 or SW480 cells were plated per well of a 24-well plate and left to attach in a 5% CO₂ incubator at 37°C for 15–20 h. At 30 min prior to TRAIL treatment, cells were preincubated in the presence of 20 µM caspase 8 inhibitor (Z-IETD-FMK), 20 µM caspase 9 inhibitor (Z-LEHD-FMK), or 20 µM each caspase 8 and 9 inhibitors. Recombinant human TRAIL (20 ng/ml, final concentration) and the anti-6xhistidine mAb (1 µg/ml, final concentration) were added to the cells in the continued presence or absence of caspase inhibitors. After 4 h of TRAIL or mock treatment in the absence or presence of caspase inhibitors, all of the drugs were removed, and cells were incubated in fresh media containing caspase inhibitors at the same concentrations for an additional 20 h, after which the inhibitors were removed, and incubation continued in fresh media. At 4 days after drug treatment, cells were stained by Coomassie Blue (22) , bright field microscopic images were taken, and colony numbers were quantified as follows. The number of colonies was counted for three fields (n = 3) of the same magnification in the case of HCT116 and two fields (n = 2) for SW480. A colony was accepted in the scoring if it contained a minimum of 6–10 cells clustered together. This definition was based on the assumption that up to three to four cell doublings would occur in 4 days, because cells had been trypsinized and later treated with different agents.

	Results

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A Caspase 9 Inhibitor, Z-LEHD-FMK, Can Protect Some Human Cancer Cell Lines from TRAIL-induced Apoptosis.
Because exposure of human cancer cells to TRAIL can lead to caspase 9 cleavage, presumably through a mechanism involving caspase 8-dependent cleavage of Bid in TRAIL-exposed cells (16) , we investigated the relative contribution of caspase 9 to TRAIL-mediated death using a caspase 9 inhibitor, Z-LEHD-FMK. Our results reveal that some human cancer cells continue to be substantially sensitive to TRAIL despite the presence of a caspase 9 inhibitor, Z-LEHD-FMK, whereas other cell lines are protected (Fig. 1A) . Thus, in these short-term apoptosis assays, the human colon cancer cell line, SW480, and the human non-small cell lung cancer cell line, H460, remained substantially sensitive to TRAIL-induced apoptosis. In the case of the H460 non-small cell lung cancer cells, there is no statistically significant difference between the apoptosis induced by TRAIL alone and by TRAIL plus the caspase 9 inhibitor. In contrast, the human colon cancer cell line, HCT116, and the human embryonic fibroblast cell line, 293, were protected completely from TRAIL-induced toxicity by the caspase 9 inhibitor. These results suggest the possibility that the signaling of death through the mitochondrial pathway may be an important component of TRAIL-induced death in some human cells. Our results suggest a different conclusion from that reached by Walczak et al. (17) , who recently reported that although Bcl2 and Bcl-X_L can delay a TRAIL-induced reduction in mitochondrial permeability transition, there was no protection offered by these inhibitors in the cell lines examined. One difference is that we used different epithelial and solid tumor cell lines, whereas Walczak et al. used lymphoma cell lines.

A Caspase 8 Inhibitor, Z-IETD-FMK, Dominant Negative FADD, or Cellular FLIP Can Protect Cells from TRAIL-induced Apoptosis.
Because a caspase 9 inhibitor, Z-LEHD-FMK, blocked TRAIL-induced apoptosis in some but not all human cancer cell lines, we investigated the dependence of TRAIL-induced apoptosis on activation of caspase 8. These studies were designed to determine whether inhibition of caspase 8 activation or enzymatic activity would protect from TRAIL-induced cell death regardless of whether caspase 9 was ultimately required for execution. Thus, if caspase 8 inhibition protects all cells from TRAIL-mediated apoptosis, these results would be consistent with the currently known signaling mechanism placing caspase 8 as the proximal initiator caspase in the TRAIL-mediated death pathway. Both dominant negative FADD and cellular FLIP-s have been shown to inhibit TRAIL-induced apoptosis (23 , 24) , and recent work has shown that both caspase 8- and FADD-deficient cells are resistant to TRAIL-induced apoptosis (25 , 26) . Our results reveal that the caspase 8 inhibitor, Z-IETD-FMK, can inhibit TRAIL-mediated killing in cells that are similarly protected or not protected by the caspase 9 inhibitor, Z-LEHD-FMK (Fig. 1A) . To further confirm these observations. we tested HCT116 and SW480 cells for whether or not they would be protected from TRAIL-induced apoptosis by DN-FADD or cFLIP-s. Our results reveal that both DN-FADD and cFLIP-s were potent inhibitors of TRAIL-induced apoptosis, regardless of the ultimate contribution of caspase 9 to the TRAIL-mediated cell death.

We further confirmed the effects of caspase inhibitors on initiator caspase cleavage and activity toward pro-caspase 3 and PARP in TRAIL-treated HCT116 and SW480 cells (Fig. 1B) . TRAIL exposure of both cell lines resulted in the depletion of the pro-caspase 8 band (Fig. 1B , Lane 1 versus 2, Lane 6 versus 7), which suggests activation of caspase 8 (the cleaved, active form of caspase 8; not shown). Pro-caspase 9 cleavage is observed to a similar extent in both cell types, and differences in its activation level do not explain the observed ability of the caspase 9 inhibitor, Z-LEHD-FMK, to inhibit cell death in HCT116 but not SW480 cells. TRAIL-induced apoptosis clearly involves procaspase 2 and procaspase 3 depletion, as well as cleavage of PARP. On the basis of these findings, it is not possible to distinguish any differences between HCT116 and SW480. However, differences become apparent on the inspection of the lanes in which caspase 9 (Z-LEHD-FMK) and caspase 2 (Z-DVADV-FMK) inhibitors have been used (Fig. 1B , Lanes 3 and 5, 8 and 10). Clearly, the caspase 9 and caspase 2 inhibitors protect procaspase 3 from cleavage in HCT116 cells but not in SW480 cells, especially at the 16-h time point. PARP cleavage is also affected, such that in HCT116 cells there is minimal processing, whereas in the SW480 cells, PARP cleavage is considerable. The caspase 8 inhibitor, Z-IETD-FMK, protects the procaspases 9, 2, and 3, and protects PARP to a similar extent in both HCT116 and SW480 cells (Fig. 1B , Lanes 4 and 9). These results are in agreement with the Annexin V-EGFP results, shown in Fig. 1A and with the long-term results shown in Fig. 2A . A somewhat surprising finding is that the caspase 2 inhibitor, Z-DVADV-FMK, gives the same pattern of results as the caspase 9 inhibitor, Z-LEHD-FMK. It is noteworthy that similar levels of pro-caspase 8 were present in untreated HCT116 and SW480 cells, and that both caspases 8 and 9 were cleaved in both cell lines. Nevertheless, the caspase 9 inhibitor, Z-LEHD-FMK, offered significant protection to the HCT116 but not the SW480 cells from TRAIL-induced procaspase 3 depletion, PARP cleavage, and ultimate cell death.

View larger version (64K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Long-term effects on cancer cells of short-term treatment with TRAIL in the presence of caspase 8 (Z-IETD-FMK) and caspase 9 (Z-LEHD-FMK) inhibitors. A, HCT116 and SW480 cells were pretreated with the inhibitors for 30 min, after which 20 ng/ml TRAIL was added, and the incubation continued for 4 h. The medium with TRAIL and the inhibitors was removed, and fresh medium with inhibitors was added and incubated for an additional 20 h. The cells were left to form colonies for 4 days, after which they were fixed and stained with Coomassie Blue. B, quantification of the results in A.

Human Hepatocytes Can Be Protected from TRAIL-induced Apoptosis by Coexposure to a Caspase 9 Inhibitor, Z-LEHD-FMK.
The observation that caspase 9 inhibition protects some but not many human cancer cells from TRAIL-induced apoptosis prompted us to investigate whether normal human hepatocytes, recently reported to be sensitive to TRAIL (11) , would be protected by the use of the caspase 9 inhibitor. We confirmed that TRAIL treatment of human hepatocytes leads to significant toxicity as demonstrated here by Annexin V-EGFP staining, indicative of early apoptosis (Fig. 3) . The results further revealed that coincubation of human liver cells with TRAIL and the caspase 9 inhibitor, Z-LEHD-FMK, leads to significant protection from TRAIL-mediated toxicity (Fig. 3B) . The results support the idea that incorporation of a caspase 9 inhibitor into a TRAIL-containing regimen may offer selective killing of some cancer cells while protecting the liver.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion Note Added in Proof REFERENCES

 
Our results provide evidence that caspase 9 may be required to a variable extent in different (TRAIL-sensitive) cells undergoing apoptosis after exposure to the cytotoxic ligand TRAIL. The underlying differences in the role that caspase 9 plays in different TRAIL-sensitive cells are not entirely clear. On exposure to TRAIL, procaspase 9 is cleaved and depleted from TRAIL-sensitive cells, regardless of whether their death could ultimately be inhibited by the caspase 9 inhibitor, Z-LEHD-FMK. It is possible that caspase 9 activity may in some cells play a more important role, perhaps in feedback amplification of the apoptotic signaling cascade, although we have not specifically investigated this possibility. It will be interesting to investigate the role of caspase 2 with regard to its contribution to TRAIL-induced apoptosis in different cells. Caspase 2, which like caspase 9 is released from the mitochondria (27) , is also activated in both HCT116 and SW480. However, the caspase 2 inhibitor, Z-VDAVD-FMK, prevents procaspase 3 and PARP cleavage in HCT116 but not in SW480 cells.

The observation that normal human liver cells can be protected from TRAIL-induced toxicity by coexposure to the caspase 9 inhibitor Z-LEHD-FMK, and the observation that some cancer cells can still be killed despite the presence of this inhibitor, suggests a strategy that can be further explored, refined, and developed for the use of systemic (or local) TRAIL in cancer therapy. Additional studies need to be performed to determine whether it will be possible to predict which cancer cells will be susceptible to the combination of TRAIL and a caspase 9 inhibitor and which cancer cells would be resistant. In this regard, we have developed a number of in vitro tests using human cancer cells (Figs. 1A and 2A) in culture to predict whether the proposed strategy of using TRAIL plus a caspase 9 inhibitor would result in cancer cell death, under conditions in which the normal human hepatocytes are protected from TRAIL. On the basis of these observations, we envision performing similar in vitro testing using viable patient tumor biopsy or surgical specimen material to attempt to predict responsiveness to the combination of TRAIL plus a caspase 9 or other caspase inhibitor. Initially such testing can be incorporated into Phase I and Phase II clinical trials with the goal that, in the future, it would be possible to stratify patients based on the predicted responsiveness of their individual tumors.

There are efforts to develop caspase inhibitors in the therapy of degenerative neurological diseases (28 , 29) . It would not be expected that a brief exposure to a caspase 9 inhibitor would have long-term consequences in terms of, for example, tumorigenicity, but these issues can be further explored in animal studies. It is clear that caspase 9 is required for apoptosis after exposure of cells to DNA-damaging agents, such as UV, -irradiation, or etoposide (30 , 31) . It would obviously not be desirable to coadminister a caspase 9 inhibitor at the same time as chemotherapy that relies on caspase 9 for killing cells.

We raised the question of whether such a strategy using a caspase inhibitor may also be applicable to Fas-induced therapy. We found that animal studies were carried out and reported showing that mice treated with a lethal injection of Fas ligand can survive if a general caspase inhibitor is coadministered (32, 33, 34) . However, in these studies, only the general caspase inhibitors Z-VAD-FMK and YVAD-CMK have been used. We propose that the caspase 9 inhibitor, Z-LEHD-FMK, can be used to differentially protect liver cells while allowing the killing of Type I tumor cells (cells that are not protected from Fas-induced apoptosis by Bcl-2) by using Fas ligand or Fas-activating Ab. The TRAIL ligand is probably more favored for development at present because of the widespread expression of its receptors and the broad sensitivities in many cancer types thus far reported. Little is known, however, about innate host- or acquired-resistance to TRAIL in cancer therapy in vivo.

In summary, our results provide an indication that suggests a novel strategy using the combination of the caspase 9 inhibitor, Z-LEHD-FMK, and TRAIL in an effort to maintain the killing effect of many cancer cell types while offering some degree of protection to the human liver. Preliminary data show that with the use of recombinant TRAIL plus the caspase 9 inhibitor Z-LEHD-FMK in coculture experiments of human hepatocytes and human colon adenocarcinoma cells, it is possible to selectively kill the cancer cells while allowing survival of the hepatocytes.⁴ In addition to the results reported herein, demonstrating the killing of colon or lung cancer cells while permitting protection of normal primary human hepatocytes by TRAIL-plus-Z-LEHD-FMK combination therapy, we have made preliminary observations with esophageal cancer and normal primary human esophageal epithelial cells in culture.⁵ These preliminary results suggest that primary human esophageal epithelial cells in culture are also sensitive to TRAIL and can be protected by Z-LEHD-FMK, whereas we have identified esophageal cancer cells that are killed by the combination of recombinant TRAIL plus Z-LEHD-FMK therapy.⁵ Thus at present the proposed strategy seems amenable to testing in patients with colon, lung, or esophageal cancer. Future studies will determine the potential range and full spectrum of tumors amenable to the proposed strategy. The same strategy may be applicable to Fas. We are currently testing this hypothesis using tumor xenograft animal models in which activation of Fas signaling leads to fulminant hepatic necrosis and death. It is also possible to improve regimens of chemotherapy and TRAIL. For example, TRAIL and the caspase 9 inhibitor can be used to kill TRAIL-sensitive tumor cells, and chemotherapy can be used on a later day of a given cycle of chemotherapy to kill TRAIL-resistant cells. Such sequential therapy may allow for initial efficient killing of tumor cells by the death receptors that rely primarily on caspase 8, as well as by subsequent exposure to chemotherapy or radiation that ultimately kill by a caspase 9-dependent mechanism. Our experiments provide evidence and proof of principle for a novel approach that can be tested in clinical trials, namely a combination of TRAIL and a caspase 9 inhibitor against cancers that are not protected by caspase 9 inhibitors against TRAIL-induced apoptosis. We are not, at this point, recommending specific doses or administration schedules that may or may not be effective in vivo. Nonetheless, the proposed strategy offers hope for the ultimate development of TRAIL as an effective and safer option for the therapy of some cancers.

	Note Added in Proof

Top ABSTRACT Introduction Materials and Methods Results Discussion Note Added in Proof REFERENCES

 
New information is emerging that not all recombinant TRAIL preparations are necessarily the same, and carefully designed experiments are being conducted to compare different preparations as well as to better understand the role of zinc ions in regulating TRAIL signaling (Donald W. Nicholson, Nature (Lond.), 407: 810–816, 2000). It is clear that the TRAIL used in the current study can cause apoptosis in a manner determined by the expression of the TRAIL receptors (Kim et al., Clin Cancer Res., 6: 335–346, 2000, and data not shown), and that in a DR4-deficient cell line, reintroduction of human TRAIL receptor DR4 enhances cell death by TRAIL (Ozoren et al., Int. J. Oncol., 16: 917–925, 2000). Moreover, the observed TRAIL-induced cell death could be substantially attenuated by DN-FADD and cellular FLIP-s (Fig. 1C) as well as the decoy TRUNDD (Meng et al., Mol. Ther., 1: 130–144, 2000), consistent with its known signaling mechanism. However, it is unclear how different TRAIL preparations could determine the downstream mechanism by which TRAIL induces death, i.e., whether death occurs by a Type I versus a Type II mechanism. It is worth noting that a different TRAIL preparation from the one used in this study was reported to be toxic to normal human astrocytes (Walczak et al., Nat. Med., 5: 157–163, 1999). Obviously the ideal TRAIL reagent to use in cancer therapy is one with broad and high efficacy but with acceptable risk from severe acute toxicity, a relevant and important issue for cancer patients. Studies using TRAIL plus a mitochondrial caspase inhibitor may provide a feasible direction within clinical trials in terms of offering a potentially acceptable risk:benefit ratio for selected patients whose tumors may be highly responsive to TRAIL therapy despite the presence of a caspase 9 or 2 inhibitor in the treatment regimen.

	ACKNOWLEDGMENTS

 
W. S. E-D. is an Assistant Investigator of the Howard Hughes Medical Institute.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ N. Ö. and K. K. contributed equally to this work.

² To whom requests for reprints should be addressed, at Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, 415 Curie Boulevard, CRB 437A, Philadelphia, PA 19104. Phone: (215) 898-9015; Fax: (215) 573-9139; Email: weldeir{at}hhmi.upenn.edu

³ The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; Ab, antibody; PARP, poly(ADP-ribose) polymerase.

⁴ N. Özören, A. D. Moscioni, and W. S. El-Deiry, unpublished observations.

⁵ K. Kim, H. Nakagawa, A. K. Rustgi, and W. S. El-Deiry. TRAIL plus a caspase 9 inhibitor induces apoptosis in human esophageal cancer but not in normal esophageal epithelial cells, manuscript in preparation.

Received 7/13/00. Accepted 9/26/00.

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Top ABSTRACT Introduction Materials and Methods Results Discussion Note Added in Proof REFERENCES

 

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