Heat Shock Protects HCT116 and H460 Cells from TRAIL-Induced Apoptosis

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1 Experimental Cell Research 281, (2002) doi: /excr Heat Shock Protects HCT116 and H460 Cells from TRAIL-Induced Apoptosis Nesrin Özören and Wafik El-Deiry 1 Departments of Medicine, Genetics, Pharmacology and Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Heat shock proteins have been shown to protect cells from a variety of stressful conditions, including hyperthermia, oxidative and DNA damage, serum withdrawal, and a variety of chemicals. HSP27, HSP70, and HSP90 have been shown to downregulate different aspects of apoptosome assembly. TRAIL is a member of the TNF family of ligands and is a promising anti-cancer agent. It has been shown to be nontoxic to most normal cell types, while it is a potent killer of many different cancer cells. TRAIL engages both the receptor-mediated (extrinsic) and the mitochondria-initiated (intrinsic) cascades. We tested whether heat shock affects TRAIL-induced apoptosis in different cancer cells. TRAIL treatment does not induce HSP27, HSP70, or HSP90 levels. Nonetheless, when treated with TRAIL for 3 h after release from heat shock, the human colon cancer cell line HCT116 is protected from apoptosis whereas the human colon cancer cell line SW480 is not. This pattern is consistent with the previously observed behavior of HCT116 as Type II cells that depend on mitochondrial signaling and SW480 as Type I, whose TRAIL-induced death is not sensitive to inhibition of caspase 9. Moreover, the failure of heat shock to protect SW480 cells is not due to a lack of HSP70 or HSP90 upregulation. HSP70 and HSP90 are induced 3 h after release from heat shock, whereas HSP27 is induced much later. Thus, the observed protective effect against TRAIL is probably due to the anti-apoptotic effects of HSP70 and HSP90. These results further illustrate interactions between TRAIL receptor signaling and the intrinsic cell death pathway and have practical implications for the potential use of TRAIL and hyperthermia in cancer therapy Elsevier Science (USA) Key Words: TRAIL; heat shock; apoptosis. INTRODUCTION 1 To whom reprint requests should be addressed. 175 TRAIL, a member of the TNF family of ligands, is a homotrimeric molecule incorporated into the cytoplasmic membrane. TRAIL can bind two death-domaincontaining receptors, namely DR4 and KILLER/DR5 causing death, as well as two decoy receptors TRID and TRUNDD [1]. TRAIL and its trimeric receptors are widely expressed throughout the human body. Upon binding of TRAIL to its two pro-apoptotic receptors, a death-inducing signaling complex (DISC) is assembled. The TRAIL-DISC contains the adaptor molecule FADD, the initiator pro-caspase 8 [2], and the regulator c-flip-l. Pro-caspases are known to have three distinct domains, the pro-domain and the large and small subunits. The close proximity of pro-caspase 8 molecules allows the autocatalytic cleavage of the small subunit of the pro-enzyme and later an active enzyme composed of two small and large subunits is formed [3]. The activation of caspase 8 initiates a cascade of cleavages of downstream effector caspases such as pro-caspases 3, 6, and 7 [4]. In addition, caspase 8 can cleave BID, a BH3-domain-only pro-apoptotic member of the BCL-2 family of apoptosis regulators [5]. The truncated form of BID translocates into mitochondria and causes cytochrome c release into the cytoplasm [6]. Cytochrome c binds the adaptor APAF-1 and the initiator pro-caspase 9, forming a complex known as the apoptosome [7]. Pro-caspase 9 can initiate apoptosis independent of caspase 8 in response to many kinds of death-inducing signals, including serum withdrawal, oxidative stress, and DNA damage. Active caspase 9 cleaves downstream effector caspases similar to caspase 8. Caspase-9-activated caspase 3 is capable of cleaving caspase 8, creating feedback amplification. The intrinsic pathway, initiated at the mitochondria, and the extrinsic receptor-mediated pathways cooperate in the cleavage of cellular targets, including fodrin, gelsolin, ICAD, PARP, etc., which dismantle the cell into apoptotic bodies later to be engulfed by neighboring healthy cells [3, 8]. The physiological function of TRAIL is still under investigation although recent studies suggest a possible role in immune surveillance. Although the ligand and its receptors are ubiquitously expressed, most normal cells are resistant to TRAIL in contrast to many cancer cell lines. This does not appear to be solely due to the presence of decoy receptors. Actually, the cells /02 $ Elsevier Science (USA) All rights reserved.

2 176 ÖZÖREN AND EL-DEIRY have a multitude of apoptosis regulators capable of inhibiting the effects of TRAIL. For example, FLIP-s is an anti-apoptotic molecule with a structure similar to that of pro-caspase 8 with a catalytically inert active site, and its ability to inhibit apoptosis is attributed to its ability to compete with pro-caspase 8 for binding to FADD. Other anti-apoptotic regulators include members of the BCL-2 family [9], such as BCL-2 and BCL- XL; members of the IAP family, IAP-1, IAP-2, XIAP [10], etc.; members of the heat shock family, such as HSP27, HSP70, and HSP90 [11, 12]. Both anti-apoptotic and pro-apoptotic (BAX, BAK) BCL-2 family members act at the outer mitochondrial membrane and are thought to regulate the release of cytochrome c [13]. IAPs are known to bind initiator and effector caspases and inhibit their enzymatic activities. However, an antagonist of IAPs, Smac/Diablo, is released from the mitochondria together with cytochrome c and it can bind IAPs, allowing caspase activity [14]. Heat shock proteins have evolved to protect cells against a variety of stressful conditions, including hyperthermia, DNA damage, oxidative stress, serum starvation, fever or inflammation, and some chemicals [12, 15]. Heat shock proteins are known to exert their protective effects through several mechanisms such as acting as chaperones to mediate proper folding of misfolded proteins and degradation of damaged proteins, as well as the recently recognized effects of inhibiting different steps of the apoptotic cascade [11]. Recently, the molecular mechanisms through which several heat shock proteins protect cells from apoptosis have been elucidated. For example, HSP70 and HSP90 interact with APAF-1 preventing efficient assembly of the apoptosome [16 18]. HSP27, on the other hand, sequesters cytochrome c from the cytoplasm, thus lowering the quantities available to form apoptosomes [19]. B-Crystallin was reported to prevent both the mitochondrial and the death receptor pathways by inhibiting the maturation of caspase 3 by binding to the p24 partially processed form of the enzyme [20]. Unlike the above-described anti-apoptotic heat shock proteins, Hsp60 and its partner HSP10 were shown to promote pro-caspase 3 maturation [21, 22]. TRAIL is a promising anti-cancer agent because of it selective toxicity to cancer cells. Although some preparations of recombinant TRAIL have been shown to be toxic to primary human hepatocytes [23 25] and primary keratinocytes [26], untagged versions appear to be less toxic [27, 28]. The use of specific antibodies to TRAIL death receptors has the potential to be developed into even safer treatment alternatives. Recently, a monoclonal antibody raised to DR5/KILLER was shown to be capable of inducing death of cancer cell lines without significant toxicity toward primary human hepatocytes [29]. Thus far, the effect of TRAIL on the heat shock or stress response and conversely the effect of heat shock on TRAIL-induced apoptosis have not been investigated. We report that TRAIL treatment does not activate the heat shock response. However, if the heat shock response is activated prior to TRAIL treatment, it confers considerable resistance to some cells. The observed effect correlates with HSP70 and HSP90 levels. Our results further illustrate regulatory links between the extrinsic cell death pathway initiated by TRAIL and cellular components that control the intrinsic apoptosome pathway. The results also have implications for the potential combined use of TRAIL and hyperthermia. MATERIALS AND METHODS Cancer cell lines. The human colon cancer cell line SW480 was obtained from the ATCC (Manassas, VA); the human non-small-cell lung cancer cell line H460 was from Dr. Stephen Baylin (John Hopkins University, Baltimore, MD), and the human colon cancer cell line HC116 was from Dr. Bert Vogelstein (John Hopkins University). HCT116 cells were grown in McCoy s 5A medium, SW480 in DMEM (high glucose), and H460 in RPMI 1640 each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All media were purchased from Gibco BRL. The cells were grown at 37 C with 5% CO 2. Western analyses. Cell lysates were run on 15% SDS PAGE and probed with the following antibodies (Ab) at the given dilutions (v/v): monoclonal mouse anti-actin Ab, 1/250 (Santa Cruz Biotechnology, Inc., Santa Cruz-CA); anti-hsp27 mouse IgG (1/1000) (SressGen Biotech. Corp., Victoria, BC, Canada); anti-hsp70 mouse IgG (1/ 1000) (SressGen); anti-hsp90 rat IgG (1/1000) (StressGen); anticaspase 3 (E-8) mouse IgG, 1/500 (Santa Cruz); and anti-parp polyclonal rabbit Ab, 1/2000 (Roche, Mannheim, Germany). TRAIL treatment. For Fig. 1, of each of the cell lines SW480, HCT116, and H460 were plated per well of 6-well plates 1 or 2 days prior to treatment. The cells were treated with 6 His-TRAIL (50 ng/ml) in the presence of the anti-6 His cross-linking antibody (1 g/ml) (both from R&D Systems, Inc., Minneapolis, MN) for 8 or 24 h. The cross-linking antibody was added to the mock-treated samples. Heat shock treatment. A total of SW480 cells were plated in T25 flasks 2 days prior to treatment for SW480 cells and 1 day or 2 days prior for HCT116 cells, in order to reach similar confluency. The cells were incubated at 42 C for 1 h after which they were transferred to a temperature of 37 C and allowed to recover for 0, 3, and 24 h. TRAIL treatment after heat shock. A total of of each of the cell lines SW480, HCT116, and H460 were plated per well of 6-well plates 2 days prior to heat shock. Cells were subjected to heat shock and left to recover for 3 h, after which they were treated with 6 His-TRAIL (50 ng/ml) in the presence of the anti-6 His crosslinking antibody (1 g/ml) (R&D Systems) for the indicated times. The cross-linking antibody was added to the mock-treated samples. Flow cytometric analysis for apoptosis. Cells treated with TRAIL for different amounts of time were collected via trypsinization, including both attached and detached cells. Live cell pellets were resuspended in a buffer containing Annexin-V EGFP and propidium iodide (PI), as recommended by the manufacturer (Annexin-V EGFP Apoptosis Detection kit, BioVision, Inc., Palo Alto, CA). The cells were analyzed using a Beckman Coulter Epics Elite Flow Cytometer.

3 HEAT SHOCK PROTECTS CELLS FROM TRAIL-INDUCED APOPTOSIS 177 FIG. 1. TRAIL treatment does not activate the heat shock response. HSP27, HSP70, and HSP90 levels were tested in SW480, HCT116, and H460 cells treated with TRAIL for 8 or 24 h. M represents mock treatment with anti-his antibody, and T represents TRAIL treatment. RESULTS TRAIL treatment of SW480, HCT116, and H460 cells does not induce HSP27, HSP70, or HSP90. Since heat shock proteins are known to be upregulated under different stressful conditions, we tested whether stress induced by TRAIL treatment might upregulate the levels of HSP27, HSP70, and HSP90. After an 8- or 24-h incubation with TRAIL HSP27, HSP70, and HSP90 levels were not induced in any of the three cell lines tested (Fig. 1). Thus, it appears that the stress of TRAIL-induced apoptosis does not lead to activation of the heat shock response. Heat shock protects HCT116 and H460 but not SW480 cells from TRAIL-induced apoptosis. We next tested the effect of heat shock on TRAIL-induced apoptosis. We induced the heat shock response by subjecting the cells to hyperthermic conditions, 42 C for1h, after which we allowed the cells to recover at 37 C for various lengths of time. HSP70 and HSP90 levels were elevated as early as 3 h after release from heat shock. HSP27 levels were slightly induced or not induced at all (Fig. 2A and 4 and data not shown) 3 h after release from heat shock. However, HSP27 levels were noted to be considerably higher at 24 h after release from heat shock (Fig. 2A). We tested whether there was protection from TRAIL-induced apoptosis after a short recovery time. After a 3-h recovery time and a 5-h TRAIL treatment there was significant protection of HCT116 but not of SW480 cells. Furthermore, when TRAIL treatment was prolonged to 15 or 16 h, apoptosis was inhibited by approximately one-third in HCT116 and H460 cells, whereas the protection was not significant in SW480 cells. It is clear that HCT116 and H460 cells were protected considerably whereas SW480 cells were not (Fig. 2B). This pattern is consistent with the previously observed behavior of HCT116 as Type II cells that depend on mitochondrial signaling and SW480 as Type I, whose TRAIL-induced death is not sensitive to inhibition of caspase 9 [24]. Heat shock slows down apoptosis downstream of procaspase 3 cleavage. We next determined whether the evident protective effect of heat shock from TRAIL toxicity could be attributed to the inhibition of caspase processing. Since pro-caspase 3 is downstream of the initiator caspases 8 and 9, any effect upstream in the cascades can be tested by looking at the level of procaspase 3 cleavage. Another downstream target of caspases is PARP. We were able to show a slight protection of pro-caspase 3 at 4 h for HCT116 cells. However, at 15 h of TRAIL treatment there was no uncleaved pro-caspase 3 in either cell line (Fig. 3). The complete cleavage of procaspase 3 at 15 h, when we see the greatest level of protection from apoptosis, was surprising. We could not detect a protection of PARP at either time point. Thus, it appears that heat shock acts on a step downstream of pro-caspase 3 cleavage and is capable of delaying cell death despite the presence of active caspase 3. HSP70 and HSP90 are upregulated in the protected HCT116 cells. We analyzed the levels of the individual heat shock proteins that may contribute to the observed protection from TRAIL-induced cell death. HSP70 and HSP90 were upregulated at 4 h (Fig. 4) TRAIL treatment, after a -h release from heat shock, in HCT116 and SW480 cells, whereas HSP27 levels remained constant, even after a 15-h incubation in the presence or presence of TRAIL (Fig. 4B). Although HSP70 and HSP90 were upregulated in both cell lines, protection from death was observed in HCT116 cells (Fig. 2). Thus, in SW480 cells the failure to protect is not due to the inability of the cells to upregulate the heat shock proteins. HSP70 and HSP90 are the likely

4 178 ÖZÖREN AND EL-DEIRY FIG. 2. Heat shock protects HCT116 and H460 but not SW480 cells from TRAIL-induced apoptosis. (A) SW480 and HCT116 cells were subjected to heat shock (HS; 42 C, 1 h) and were allowed to recover (37 C) for 0, 3, and 24 h. (B) Cells released from heat shock for 3 h were treated with TRAIL (50 ng/ml) for the indicated times. Apoptosis was assessed using the Annexin-V EGFP assay. The percentage of cell death is the sum of Annexin-V( )/PI( ) and Annexin-V( )/PI( ) cells. Experiments were carried out in triplicates, SEM shown. negative effectors of TRAIL-induced apoptosis after heat shock in HCT116 cells and probably other cells that behave as Type II in response to TRAIL exposure [24]. DISCUSSION The heat shock response is activated in response to various stresses including hyperthermia, oxidative damage, DNA damage, serum withdrawal, fever or inflammation, and various chemicals. Thus far, the effect of TRAIL treatment on the heat shock response and vice versa, the effect of heat shock on TRAILinduced cell killing, has not been reported. Our study shows that the stress inflicted by TRAIL treatment does not result in the upregulation of the three major anti-apoptotic heat shock proteins, namely HSP27, HSP70, and HSP90. When the heat shock response was activated using hyperthermia and the cells were allowed to recover for 3 h, HCT116 and H460 but not SW480 cells were protected from TRAIL-induced apoptosis. After a 3-h recovery period, HSP70 and HSP90 are upregulated in both HCT116 and SW480 cells (Fig. 4), and yet SW480 cells are not protected (Fig. 2B). One explanation may be the differing levels of dependence on the mitochondrial amplification loop. We have shown that inhibition of caspase 9 protects HCT116 cells from TRAIL-induced apoptosis completely, whereas the protection is partial in SW480 cells [24]. Because heat shock proteins regulate apoptosis by opposing the formation of the apoptosome efficiently, it could be suggested that SW480 are not protected because they are less dependent on the mitochondria (they die through a Type I pathway in response to TRAIL;[24]). Since TRAIL and FAS share many similarities in their molecular mechanisms of apoptosis induction we searched the literature for reports on the effect of heat shock on FAS-induced cell death and we found conflicting reports. In human Jurkat T cells heat shock was found to protect from CD95L (FAS-L)-induced apoptosis [30] and yet in another study the same cells were not protected against anti-fas-ab-induced cell death. It has been reported that human HSP27 can prevent

5 HEAT SHOCK PROTECTS CELLS FROM TRAIL-INDUCED APOPTOSIS 179 inhibit cytochrome c release and procaspase 3 activation but inhibited apoptotic changes of the nuclear morphology and the cells returned to normal growth after the lethal stimuli [33]. This group used TNF, staurosporine, and doxorubicin to induce apoptosis in intact ME-180 human cervical carcinoma cells and WEHI-S murine fibrosarcoma cells. The reasons for the different outcomes are not clear; however, different apoptotic stimuli and cell lines were used in the above studies. We carried out our experiments with intact cells using TRAIL to induce apoptosis and our data suggest that apoptosis can be inhibited, even in the presence of active caspase 3. Alternatively, the inability of heat shock to prevent caspase 3 activation may be due to the fact that, in the HCT116 and SW480 colon FIG. 3. Heat shock does not prevent pro-caspase 3 and PARP cleavage in TRAIL-treated cells. Cells subjected to heat shock (42 C, 1 h) and 3 h release (37 C) were treated with TRAIL (50 ng/ml) for 4 or 15 h. Pro-caspase 3 and PARP levels were evaluated in equally loaded samples. FAS/APO-1 induced apoptosis in murine L929 cells [31]. HSP70 was found to protect against etoposide but not against anti-fas-ab [17]. It is clear that further investigation is necessary to elucidate these conflicting findings. One interesting aspect of our study is the finding that even if there is active caspase 3 in the cells, apoptosis can be inhibited. We show that under the conditions where apoptosis is affected, pro-caspase 3 and PARP are still depleted. Since our apoptosis assay measures phosphoserine externalization and propidium iodide uptake, it could be suggested that heat shock acts downstream of caspase 3. The issue of whether heat shock proteins act upstream or downstream of caspase 3 activation is also controversial. HSP70 was found to inhibit pro-caspase 3 processing upon the addition of cytochrome c to extracts from 293T human embryonic kidney cells overexpressing HSP70. However, the addition of recombinant caspase 8 to the same extracts resulted in procaspase 3 processing despite HSP70 [17]. In another study, HSP70 was found to inhibit apoptosis downstream of cytochrome c release but upstream of caspase 3 activation in cytosolic extracts from U937 human leukemia cells overexpressing HSP70, where apoptosis was activated by the addition of datp [32]. On the other hand, Jaattela et al. reported that HSP70 expression did not FIG. 4. HSP70 and HSP90 but not HSP27 levels are upregulated in the cells treated with TRAIL after heat shock. (A) The levels of HSP27, HSP70, and HSP90 were evaluated after 4 h TRAIL (50 ng/ml) treatment following a 3-h release from heat shock. (B) HSP27 is not upregulated even after a 15-h TRAIL treatment, after a 3-h release from heat shock.

6 180 ÖZÖREN AND EL-DEIRY carcinoma cells, both the intrinsic and the extrinsic pathways are activated. Thus, blocking the mitochondrial branch does not influence the effector caspases activated by caspase 8 at the TRAIL-DISC. This notion is further supported by the intermediate protective effect observed in H460 cells. Since heat shock proteins affect apoptosome assembly, the extrinsic pathway is apparently still operational. Heat shock protein levels were correlated with clinical outcome in prostate cancer. High levels of HSP27 re-expression in invasive prostatic carcinoma after a lack of expression in the early prostatic neoplasia stage were found to be indicative of poor clinical outcome [34]. Since TRAIL is a potential anti-cancer therapeutic, the effect different heat shock proteins have on TRAIL-induced apoptosis must be elucidated. We were able to show that heat shock can prevent cell death in some but not all cells. 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