Hyperthermia enhances tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells

Abstract

Purpose: This study investigated whether hyperthermia can enhance TRAIL-induced apoptotic death.

Methods: Human prostate adenocarcinoma DU-145, human pancreatic carcinoma MIA PaCa-2 and BxPC-3, human colon fibroblast CCD-33Co and rat prostate endothelial YPEN-1 cells were treated with various concentrations of TRAIL (0–200 ng ml⁻¹) with hyperthermia (40–42°C).

Results: It was observed in human cancer cells, but not in normal cells, that TRAIL induced apoptotic death and also that hyperthermia (40–42°C) promoted TRAIL-induced apoptotic death. Enhancement of TRAIL-mediated apoptosis by hyperthermia was detected by an increase in PARP cleavage, the hallmark feature of apoptosis, as well as by activation of caspases. There were no significant changes in the intra-cellular levels of death receptors (DRs), decoy receptors (DcRs) and anti-apoptotic proteins. Interestingly, data from in vitro enzyme kinetics assay demonstrated that hyperthermia promoted caspase enzyme activity.

Conclusions: These data suggest that cancer cells are more susceptible to TRAIL in the condition of hyperthermia (40–42°C). The promotion of caspase enzyme activity by hyperthermia may be responsible for enhancement of TRAIL-induced apoptotic death.

• TRAIL
• apoptosis
• hyperthermia
• CX-1
• TUNEL
• PARP-1
• caspase-3
• caspase-8
• caspase-9

Abbreviations

DcR1:	=	decoy receptor 1
DcR2:	=	decoy receptor 2
DR4:	=	death receptor 4
DR5:	=	death receptor 5
DTT:	=	dithiothreitol
FADD:	=	Fas-associated death domain
FasL:	=	Fas ligand
PARP:	=	Poly-ADP Ribose Polymerase
FLICE:	=	Fas-associated death domain-like interleukin-1β-converting enzyme
FLIP:	=	FLICE inhibitory protein
IAP:	=	inhibitor of apoptosis
PAGE:	=	polyacrylamide gel electrophoresis
PARP:	=	poly (ADP-ribose) polymerase
PBS:	=	phosphate-buffered saline
PDK-1:	=	phosphoinositide-dependent kinase-1
PI3K:	=	phosphatidylinositol 3-kinase
PP1:	=	protein phosphatase 1
PTEN:	=	phosphatase and tensin homologue deleted on chromosome 10
SDS:	=	sodium dodecyl sulphate
TNF:	=	tumour necrosis factor
TRAIL:	=	tumour necrosis factor-related apoptosis-inducing ligand

Introduction

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), a 281-amino-acid protein, is a type II integral membrane protein belonging to the tumour necrosis factor (TNF) family, which includes Fas ligand (FasL) and TNF. The C-terminal extra-cellular region of TRAIL (amino acids 114–281) exhibits a homotrimeric sub-unit structure [1] and its function is to induce apoptosis of tumour cells. The mechanism of TRAIL-induced apoptosis is initiated when TRAIL binds to death receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5) which induces the apoptotic signal. Both DR4 and DR5 contain a cytoplasmic death domain, which is required for TRAIL receptor-induced apoptosis. TRAIL also binds to decoy receptors (DcR1, DcR2), which results in inhibition of TRAIL signalling [2–8]. Recent studies revealed that inhibition of TRAIL signalling by DcR2 critically depends on its association with DR5 via the NH₂-terminal pre-ligand assembly domain overlapping the first partial cysteine-rich domain of both receptors [9]. These observations suggest that DcR2 is a regulatory rather than decoy receptor. Nevertheless, the relative resistance of normal cells to the apoptotic inducing effects of TRAIL has been explained by the presence of large numbers of the decoy receptors on normal cells [10], [11]. Recently, this hypothesis has been challenged based on the results showing poor correlations between DR4, DR5 and DcR1 expression and sensitivity to TRAIL-induced apoptosis in normal and cancerous breast cell lines [12] and melanoma cell lines [13]. This discrepancy indicates that other factors such as death inhibitors including FLICE-inhibitory protein (FLIP) [13], Fas-associated protein (FAP-1) [14], Bcl-2 [15], Bcl-X_L [15], Bruton's tyrosine kinase (BTK) [16], silencer of death domain (SODD) [17], toso [18], inhibitor of apoptosis (IAP) [19], X-linked inhibitor of apoptosis (XIAP) [20] and survivin [21] may be responsible for the differential apoptotic effect of TRAIL.

As mentioned above, TRAIL has been shown to induce apoptosis in a broad range of cancer cell types but not in normal cells and tissues, suggesting that it could be a potential therapeutic agent for the treatment of cancer [22–24]. However, many tumour cells have been shown to be resistant to TRAIL [25], [26]. Several researchers have reported that TRAIL resistance can be overcome by various sensitizing agents such as chemotherapeutic drugs [12], [13], [27–29], ionizing radiation [30], cytokines [31] and matrix metalloprotease inhibitors [32]. It has also been demonstrated that tumour micro-environments such as low extra-cellular pH [33], low glucos [34], [35] and low oxygen tensions [36] augment TRAIL cytotoxicity. This study investigated whether hyperthermia also promotes TRAIL-induced cytotoxicity.

Hyperthermia, that is temperature above normal (37°C), has been successfully used to enhance the effectiveness of various forms of anti-cancer treatment, including chemotherapy and radiation therapy [37–39]. Randomized clinical trials showed that complete response rate with radiotherapy plus hyperthermia was significantly better than that with radiotherapy alo [40], [41]. A phase II study also demonstrated that chemotherapy combined with hyperthermia improved local tumour control and overall survival in comparison with chemotherapy alo [42], [43]. However, several problems still remain to be solved to optimize the efficiency of hyperthermia in cancer therapy. Conventional studies have demonstrated that heating non-superficial tumours above 42°C is technically difficult and commonly leads to pai [44], [45]. This problem can be overcome by a multi-modality treatment strategy including the use of sub-lethal temperatures[41], [43], [46]. This study observed that hyperthermia (41–42°C) effectively enhances TRAIL-induced cytotoxicity by promoting caspase activity. It is believed that hyperthermia in combination with TRAIL can be used as an adjunctive therapy for cancer treatments such as radiotherapy and chemotherapy.

Materials and methods

Cell culture and survival assay

Human prostate adenocarcinoma DU-145, human pancreatic carcinoma MIA PaCa-2 and BxPC-3 and rat prostate endothelia YPEN-1 cells were cultured in DMEM medium (Gibco BRL, Gaithersburg, MD) containing 10% foetal bovine serum (HyClone, Logan, UT) and 26 mM sodium bicarbonate for monolayer cell culture. Human colorectal carcinoma CX-1 cells were cultured in RPMI-1640 medium (Gibco BRL) containing 10% foetal bovine serum. Human colon fibroblast CCD-33Co cells were cultured in Eagle's Minimal Essential medium containing 10% foetal bovine serum, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine and 1.5 g L⁻¹ sodium bicarbonate. The dishes containing cells were kept in a 37°C humidified incubator with 5% CO₂. One or 2 days prior to the experiment, cells were plated into 60-mm dishes. For trypan blue exclusion assay [47], trypsinized cells were pelleted and resuspended in 0.2 ml of medium, 0.5 ml of 0.4% trypan blue solution and 0.3 ml of phosphate-buffered saline solution (PBS). The samples were mixed thoroughly, incubated at room temperature for 15 min and examined under a light microscope. At least 300 cells were counted for each survival determination. For colony formation assay, the 60-mm Petri dishes containing monolayers of asynchronous cells were trypsinized with ice cold 0.05% trypsin and EDTA (0.1 g l⁻¹) in PBS. After trypsinization, the cells were resuspended in 5 ml of DMEM or RPMI-1640 medium containing 10% foetal bovine serum used as a trypsin inhibitor. Cells were counted and appropriate dilutions were made. The appropriate number of cells were plated. After 10 days of incubation at 37°C, colonies were stained and counted.

Production of recombinant TRAIL

A human TRAIL cDNA fragment (amino acids 114–281) obtained by RT-PCR was cloned into a pET-23d (Novagen, Madison, WI) plasmid and His-tagged TRAIL protein was purified using the Qiagen express protein purification system (Qiagen, Valencia, CA).

Hyperthermia treatment

Cells cultured in 60-mm dishes were sealed with parafilm and were placed in a circulating water bath (Heto, Thomas Scientific, Denmark) which was maintained within ±0.02°C of the desired temperature.

Morphological evaluation

Approximately 5 × 10⁵ cells were plated into 60-mm dishes overnight. Cells were treated with TRAIL and/or hyperthermia and then analysed by phase contrast microscopy for signs of apoptosis.

TUNEL assay

For detection of apoptosis by the TUNEL method, cells were plated in slide chambers. After treatment, cells were fixed with 4% paraformaldehyde in PBS. Cells were washed once, permeabilized by incubating with 100 µl of 0.1% Triton X-100 and 0.1% sodium citrate and then washed twice in PBS. The TUNEL reaction was carried out with 50 µl of TUNEL reaction mixture (450 µl of label solution/50 µl of enzyme solution; AP cell death detection kit, Roche, Germany). After washing three times with PBS, 50 µl of converter-AP was added on a sample. Thirty minutes after incubation in a humidified chamber at 37°C, each 100 µl of substrate (NBT/BCIP, Roche) was added. After 10 min incubation in the dark, cells were subjected to washing and were examined under a microscope.

Flow cytometry

Cells were pelleted, washed with PBS and resuspended in 200 µl of staining buffer containing fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) according to the manufacturer's instructions (BD Pharmingen, San Diego, CA). After 15 min of incubation, 300 µl of sorting buffer was added and analysis was performed using the FACScan flow cytometer (Beckman Coulter, Inc., Hialeah, FL). Results were analysed with CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Antibodies

Rabbit polyclonal anti-caspase-3 antibody was purchased from Santa Cruz (Santa Cruz, CA). Anti-DR4, anti-DR-5, anti-DcR1 and anti-DcR2 antibodies were from ProSci (Poway, CA). Anti-cIAP-1 and anti-cIAP-2 antibodies were from R&D Systems (Minneapolis, MN). Anti-phospho-Akt, anti-Akt, anti-caspase-8, anti-FLIP (C-term: 447–464), anti-FLIP γ/δ and anti-FLIP-L antibodies were from Cell Signaling (Beverly, MA). Monoclonal antibodies were purchased from each of the following companies: anti-Bcl-2 and anti-Bcl-XL antibodies from Santa Cruz, anti-caspase-9 antibody from Upstate Biotechnology (Lake Placid, NY), anti-PARP antibody from Biomol Research Laboratory (Plymouth Meeting, PA) and anti-actin antibody from ICN (Costa Mesa, CA).

Protein extracts and polyacrylamide gel electrophoresis (PAGE)

Cells were lysed with 1 × Laemmli lysis buffer (2.4 M glycerol, 0.14 M Tris, pH 6.8, 0.21 M sodium dodecyl sulphate, 0.3 mM bromophenol blue) and boiled for 10 min. Protein content was measured with BCA Protein Assay Reagent (Pierce, Rockford, IL). The samples were diluted with 1 × lysis buffer containing 1.28 M β-mercaptoethanol and equal amounts of protein were loaded on 8–12% sodium dodecyl sulphate (SDS)-polyacrylamide gels. SDS-PAGE analysis was performed according to Laemmli [48] using a Hoefer gel apparatus.

Immunoblot analysis

Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% non-fat dry milk in PBS-Tween-20 (0.1%, v/v) at 4°C overnight. The membrane was incubated with primary antibody (diluted according to the manufacturer's instructions) for 2 h. Horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody. Immunoreactive protein was visualized by the chemiluminescence protocol (ECL, Amersham, Arlington Heights, IL).

In vitro caspase activity assays

Activities of caspase-8, caspase-9 and caspase-3 were measured by spectrophotometric detection of the chromophore p-nitroalanine (pNA) at 405 nm (Chemicon, Temecula, CA). The assay was performed by the manufacturer's instructions. Briefly, CX-1 cells treated or untreated with TRAIL were harvested and lysed with 1X caspase lysis buffer. The cell lysates were chilled in ice for 10 min and the insoluble fraction was removed by centrifugation of 10 000 g, 5 min at 4°C. Supernatants were used for further caspase assay. Total protein was measured by BCA protein assay kit (Pierce). To measure caspases’ activity, 50 µg of total protein was used for each assay.

Results

Effect of hyperthermia on TRAIL-induced cytotoxicity

To study the effect of hyperthermia on TRAIL-induced cytotoxicity, human colorectal carcinoma CX-1 cells were treated with various concentrations of TRAIL (0–200 ng ml⁻¹) for 4 h at various temperatures (37–42°C). Data from morphological assay and trypan blue exclusion assay show that no or minimal cytotoxicity was observed during hyperthermia (40–42°C) alone (Figures 1a and b). However, unlike physiological death, colony formation assay shows that hyperthermia at 42°C for 4 h induced clonogenic cell death (Figure 1c). Additionally, TRAIL-induced cytotoxicity was promoted by hyperthermia (40–42°C). Figure 1(b) clearly shows that the augmentation of TRAIL-induced cytotoxicity was dependent upon heating temperatures. These observations were consistent with morphological features (Figure 1a). Most cells underwent apoptosis during TRAIL treatment in combination with hyperthermia as shown by cell surface blebbing and formation of apoptotic bodies (Figure 1a). An increase in the number of rounded cells and detached cells was observed during treatment with TRAIL in combination with hyperthermia. Similar results were observed with TUNEL assay (Figure 1d) and flow cytometric assay (Figure 1e). Data from TUNEL and flow cytometric assays show that apoptotic death occurred during treatment with TRAIL and hyperthermia increased TRAIL-induced apoptotic death.

Figure 1. Effect of hyperthermia (40–42°C) on TRAIL-induced apoptosis in human colorectal carcinoma CX-1 cells. (a) Cells were treated with 200 ng ml⁻¹ TRAIL for 4 h at 37°C or 42°C. The morphological features were analysed with a phase-contrast microscope. (b) Cells were treated with various concentrations of TRAIL (0–200 ng ml⁻¹) for 4 h at various temperatures (37–42°C). Cell viability was determined by the trypan blue exclusion assay. Error bars represent standard error of the mean (SEM) from three separate experiments. Asterisks indicate values which are different from the respective control (t-test, p < o.05). (c) Cells were treated with 50 ng ml⁻¹ TRAIL for 4 h at 37 or 42°C. Cell survival was determined by the colony formation assay. Error bars represent standard error of the mean (SEM) from three separate experiments. Asterisks indicate values which are different from the respective control (t-test, p < 0.05). (d) Cells were treated with TRAIL (200 ng ml⁻¹) for 2 h at 37°C or 42°C. After treatment, apoptosis was detected by the TUNEL assay. (e) Cells were treated with TRAIL (50 ng ml⁻¹) for 4 h at 37°C or 42°C. After treatment, apoptosis was detected by the flow cytometric assay. (f) Proteolytic cleavage of PARP and activation of caspases were assayed by Western blot analyses. Cells were treated for 4 h with various concentrations of TRAIL at various temperatures. Cell lysates were harvested and subjected to immunoblotting for caspase-8, caspase-9, caspase-3 or PARP. Antibody against caspase-8 detects inactive form (55/54 kDa) and cleaved intermediates (41, 43 kDa). Anti-caspase-9 antibody detects both inactive form (48 kDa) and cleaved intermediate (37 kDa). Anti-caspase-3 antibody detects inactive form (32 kDa) and cleaved active form (17 kDa). Immunoblots of PARP show the 116 kDa PARP and the 85 kDa apoptosis-related cleavage fragment. Actin was used to confirm the equal amount of proteins loaded in each lane.

Additional studies were designed to examine whether the combination of hyperthermia and TRAIL treatment enhances poly (ADP-ribose) polymerase (PARP) cleavage, the hallmark feature of apoptosis. PARP (116 kDa) was cleaved yielding a characteristic 85 kDa fragment in the presence of TRAIL (10–200 ng ml⁻¹). This cleavage was enhanced by hyperthermia, in particular heating at 42°C (Figure 1f). It is well known that TRAIL-induced apoptosis is mediated through a caspase cascade. To examine whether hyperthermia enhances TRAIL-induced apoptosis through activation of caspases, several caspases known to be involved in TRAIL-induced apoptosis were examined. Figure 1(f) shows that hyperthermia enhanced TRAIL induced caspase-8 activation. Western blot analysis shows that procaspase-8 (55/54 kDa) cleavage to intermediate (43/41 kDa) forms was enhanced with increasing temperature in the presence of TRAIL. Hyperthermia enhanced the proteolytic processing of procaspase-9 (48 kDa) into its active form (37 kDa). Hyperthermia also increased TRAIL-induced caspase-3 activation. Western blot analysis shows that procaspase-3 (32 kDa), the pre-cursor form of caspase-3, was cleaved to active form (17 and 12 kDa) in the presence of TRAIL. The combined treatment with TRAIL and hyperthermia increased the level of active form, in particular 12 kDa.

These studies were extended to investigate the time course of apoptotic death during treatment with TRAIL in combination with hyperthermia (42°C). Figure 2 shows that apoptotic death gradually increased when the treatment time was increased. A maximal cytotoxicity occurred within 3 h. Similar results were observed for PARP cleavage as well as activation of caspases.

Figure 2. Time course of apoptotic cell death during treatment with TRAIL in the conditions of normothermia and hyperthermia. (a) CX-1 cells were treated with 50 ng ml⁻¹ TRAIL for various times (0.5–4 h) at 37°C or 42°C. The morphological features were analysed with a phase-contrast microscope. Con, untreated unheated control cells. (b) Cell viability was determined by the trypan blue exclusion assay. Error bars represent standard error of the mean (SEM) from three separate experiments. Asterisks indicate values which are different from the respective control (t-test, p < 0.05). (c) Time course of PARP cleavage and activation of caspases were assayed by Western blot analyses as described in Figure 1.

To examine whether these observations are unique to CX-1 cells, human prostate adenocarcinoma DU-145, human pancreatic carcinoma MIA PaCa-2 and BxPC-3, human colon fibroblast CCD-33Co and rat prostate endothelia YPEN-1 cells were also employed. Figures 3 and 4 show that the combined treatment of TRAIL and hyperthermia resulted in an increase in cell death and PARP cleavage as well as the activation of caspases in tumour cells but not in normal CCD-33Co and YPEN-1 cells. These results suggest that the response of normal cells to TRAIL in combination with hyperthermia differs from that of tumour cells.

Figure 3. Effect of hyperthermia on TRAIL-induced apoptosis in other cell lines: MIA PaCa-2, BxPC-3 and DU-145 cells. (a) MIA PaCa-2 and BxPC-3 cells were heated at 40°C or 42°C for 4 h in the presence or absence of 50 ng ml⁻¹ TRAIL. Cell survival was determined by the trypan blue exclusion assay. Error bars represent standard error of the mean (SEM) from three separate experiments. Asterisks indicate values which are different from the respective control (t-test, p < 0.05). (b, c) Cell lysates were subjected to immunoblotting for PARP and caspases as described in Figure 1.

Figure 4. Effect of hyperthermia in combination with TRAIL on cell morphology, proteolytic cleavage of PARP and activation of caspases in normal cell lines CCD-33Co and YPEN-1. (a) CCD-33Co cells were heated at 42°C for 4 h in the presence or absence of 50 ng ml⁻¹ TRAIL. The morphological features were analysed with a phase-contrast microscope. (b, c) CCD-33Co cells or YPEN-1 cells were heated at 42°C for 4 h in the presence or absence of 50 ng ml⁻¹ TRAIL. Cell lysates were subjected to immunoblotting for PARP and caspases as described in Figure 1.

Effect of hyperthermia on the TRAIL receptor family

It is well known that TRAIL-induced apoptotic signals are triggered by interaction with two death receptors (DR4 and DR5). Such signals may be blocked by antagonistic decoy receptors (DcR1 and DcR2). Previous studies also demonstrate that increased DR5 levels induced by genotoxic agents are responsible for increasing TRAIL cytotoxicity. Thus, it was examined whether treatment with TRAIL in combination with hyperthermia affects the level of TRAIL receptors. Data from western blot analysis revealed that the combined treatment did not significantly alter the total cellular levels of death receptors DR4 and DR5 as well as decoy receptors DcR1 and DcR2 (Figure 5).

Figure 5. Intra-cellular levels of TRAIL receptors during treatment with TRAIL in the conditions of normothermia and hyperthermia. CX-1 cells were treated for 4 h with various concentrations of TRAIL (0–200 ng ml⁻¹) in the indicated temperatures. To measure the levels of death receptors and decoy receptors during treatment with TRAIL in normothermia and hyperthermia, equal amounts of protein (20 µg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted as described in Materials and methods. Actin was shown as an internal standard.

Effect of hyperthermia and TRAIL on anti-apoptotic proteins

It is well known that reduction of intra-cellular anti-apoptotic molecules such as FLIP, IAP-1, IAP-2, Bcl-2 and Bcl-X_L sensitizes TRAIL-resistant cancer cells to TRAIL. Thus, this study examined whether changes in the amounts of anti-apoptotic proteins are associated with the promotion by hyperthermia of apoptosis by TRAIL. Data from western blot analysis reveal that the combined treatment did not significantly alter the levels of FLIP_L, FLIP_S, IAP-1, IAP-2 and Bcl-X_L (Figure 6). Interestingly, there is no detectable level of Bcl-2 in CX-1 cells.

Figure 6. Intra-cellular levels of anti-apoptotic proteins during treatment with TRAIL in the conditions of normothermia and hyperthermia. CX-1 cells were treated for 4 h with various concentrations of TRAIL (0–200 ng ml⁻¹) in the indicated temperatures. To determine the intra-cellular levels of anti-apoptotic proteins during treatment with TRAIL in normothermia and hyperthermia, equal amounts of protein (20 µg) were separated and immunoblotted as described in Materials and methods. Actin was shown as an internal standard.

Effect of hyperthermia on caspase activity

TRAIL induced cell death is mediated through caspase cascades. So, it was hypothesized that caspase enzyme activity is thermodynamically enhanced in hyperthermic conditions and hyperthermia-enhanced caspase enzyme activity is responsible for enhancement of TRAIL-induced apoptotic death. To test this possibility, CX-1 cells were treated with TRAIL, cell lysates were harvested and then caspase activity was measured during hyperthermia (42°C). Data from in vitro enzyme kinetics assay in Figure 7 demonstrated that hyperthermia significantly promoted caspase enzyme activity.

Figure 7. Effects of hyperthermia on caspase activity. CX-1 cells were treated with or without 200 ng ml⁻¹ of TRAIL for 2 h and the cell lysates were prepared for caspase activity. Cell lysates was subjected to reaction at 37°C or 42°C for 2 h. U, untreated cell lysates. T, TRAIL treated cell lysates. Error bars represent standard error of the mean (SEM) from three separate experiments. Asterisks indicate values which are different from the respective control (t-test, p < 0.05).

Discussion

TRAIL induces apoptosis in a wide variety of tumour cells, but does not cause toxicity to most normal cells. Pre-clinical studies in mice and primates have also shown that administration of TRAIL can induce apoptosis in human tumours, but no cytotoxicity to normal organs or tissue [23]. Recent phase 1 and 2 clinical trials using agonistic monoclonal antibodies which bind to human TRAIL receptors DR4 and DR5 have provided encouraging results for cancer therapy [49].

Previous studies have demonstrated that hyperthermia induces improvement of tumour oxygenation and increases responses of tumours to radiotherapy and chemotherapy [50–53]. This study reveals that hyperthermia (40–42°C) promotes TRAIL-induced apoptotic death by facilitating caspase activity. The observations are similar to previous reports which reveal the synergistic effect of hyperthermia on TNF- and/or interferon-γ-induced cytotoxicity [54–56]. However, these observations are somewhat different from a previous report that heat shock inhibits rather than promotes TRAIL-induced apoptosis [57]. In the previous report, cells were heated prior to TRAIL treatment, so the resistance to TRAIL may have been due to the synthesis of anti-apoptotic molecules such as HSP70 and HSP90. It is well known that HSP70 and HSP90 interact with Apaf-1 to prevent efficient assembly of the apoptosome [58–61] or antagonize the caspase-independent death effector apoptosis inducing factor (AIF) [62]. Nonetheless, the data clearly demonstrated that simultaneous TRAIL treatment and hyperthermia promotes apoptotic death by enhancing caspase activation.

Several researchers have shown that an increase in DR5 gene expression or decrease in FLIP expression in response to genotoxic stress is responsible for the synergistic effect of ionizing agents or chemotherapeutic agents on TRAIL [12], [13], [27], [30], [63]. This study revealed that hyperthermia-enhanced TRAIL cytotoxicity is not due to alteration of TRAIL receptor levels (Figure 5) or anti-apoptotic protein levels (Figure 6).

Although this study has demonstrated that caspase enzyme activity is thermodynamically enhanced in hyperthermic conditions, one cannot rule out other possibilities. One possibility is that apoptotic signalling via a mitochondria-dependent pathway is also enhanced during hyperthermia. This is based on findings that over-expression of Bcl-2 gene inhibits mitochondria-dependent apoptosis pathways and that over-expression of Bcl-2 gene also inhibits the enhancing effect of hyperthermia, PARP cleavage and caspase activation (data not shown). It is also well known that cytochrome c release from mitochondria activates various caspases during apoptosis [64–66]. It is possible that hyperthermia promotes cytochrome c release from mitochondria and the inclusion of the involvement of the mitochondria-dependent pathway may elucidate the enhancement of TRAIL-induced cytotoxicity by hyperthermia. It is believed that the model presented in this article provides a framework for future studies.

Acknowledgements

This work was supported by the following grants: NCI grants CA95191, CA96989 and CA121395, DOD prostate program fund (PC020530 and PC040833) and Susan G. Komen Breast Cancer Foundation (BCTR60306).