Bax-mediated mitochondrial membrane permeabilization after heat treatment is caspase-2 dependent

Abstract

Heat-induced apoptosis proceeds via mitochondria by permeabilization of the outer mitochondrial membrane (MOMP), resulting in the release of cytochrome c. This essential step is mediated by Bcl-2 family proteins, such as Bax. Recently, caspase-2 was assigned a prominent role in regulating Bax. Therefore, we studied the initiation of heat-induced apoptosis by monitoring Bcl-2 family members and the release of cytochrome c with or without caspase-2 inhibition. Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1) were exposed to heat treatment and/or X-radiation. Expression and localization of Bax and Bcl-2 proteins was investigated by flow cytometry (FCM) and confocal microscopy respectively. Cytochrome c release was measured with FCM as evidence for MOMP. In addition, the role of caspase-2 in heat- and radiation-induced apoptosis was assessed using the specific caspase-2 inhibitor zVDVAD-fmk. Here we present evidence that heat treatment, and not irradiation, increases intracellular Bax protein expression and subsequently stimulates MOMP, resulting in the release of cytochrome c. Furthermore, by selective blocking of caspase-2 using zVDVAD-fmk less Bax was expressed and subsequently a significant decrease in cytochrome c release was observed. In conclusion, heat treatment of hematopoietic cells does require caspase-2 activation for the initiation of Bax-mediated MOMP.

• Apoptosis
• heat treatment
• Bax
• Bcl-2
• caspase-2

Introduction

Various cancer therapy related inducers, such as hyperthermia and radiotherapy lead to apoptosis. However, the precise mechanisms how apoptosis is initiated by these inducers are still not completely elucidated. In order to map out therapeutic strategies to improve the anti-tumor effect, it is of primary importance to know how these modalities induce apoptosis. This is especially the case when therapies are used in a multimodal way such as hyperthermia in conjunction with radiotherapy.

The Bcl-2 family members and antagonists Bcl-2 and Bax are essential in inducing the apoptotic process by regulating the mitochondrial outer membrane permeabilization (MOMP) [1–3]. Once activated, the multidomain BH3 pro-apoptotic proteins Bax and Bak permeabilize the mitochondrial outer membrane in order to release cytochrome c into the cytosol [4], [5]. Cytochrome c, which normally resides exclusively in the intermembrane space of mitochondria, activates caspase-9 through Apaf-1. Caspase-9 in turn cleaves and thereby activates executioner caspases, which in turn orchestrate apoptosis.

Anti-apoptotic Bcl-2 suppresses apoptosis whereas pro-apoptotic Bax counteracts Bcl-2 function to promote apoptosis. These proteins exert their activity via interactions, such as homodimerization and heterodimerization via the Bcl-2 Homology (BH) domains. Based on these interactions, the so-called rheostat model suggests that the balance between pro- and anti-apoptotic Bcl-2 family members determines cell sensitivity to apoptotic triggers [6].

How the Bcl-2 family proteins exactly regulate MOMP is unclear. There is no consensus yet on the order in which actions occur. However, in the emerging view BH3-only proteins appear to act upstream of the multidomain proteins (Bax and Bcl-2) to initiate apoptosis [7], [8]. Recently, the BH3-only protein Bid has been shown to be cleaved into truncated Bid (tBid) at the same site not only by caspase-8 in the extrinsic pathway of apoptosis, but also by caspase-2 during intracellular stimulation such as hyperthermia [9–11]. This finding indicates an important function of caspase-2 in stimulating MOMP through the cleavage of Bid after intracellular activation. Although caspase-2 was the second caspase to be identified and the most evolutionary conserved [12], there is a considerable debate as to whether it is an initiator or effector caspase [13], [14]. Findings that caspase-2 resides upstream of mitochondria show that it appears to function as an initiator caspase. Recently, a role for caspase-2 as initiator caspase in heat-induced apoptosis has been suggested by Tu et al. [15].

In our previous study it was indicated that the commitment to apoptosis in HL60 promyelocytic cells after heat treatment starts with the mitochondrial membrane transition and is mainly executed in a caspase-dependent pathway [16]. In addition, our results suggested a key role for caspase-2 in heat-induced apoptosis. In the present study we investigated the regulation of MOMP by Bcl-2 and Bax and the subsequent cytochrome c release in more detail in several leukemic cell lines after heat treatment and/or X-irradiation. Furthermore, by specific inhibition of caspase-2 with zVDVAD-fmk the role of this enzyme in Bax activation and cytochrome c release was studied.

Materials and methods

Cell cultures

The acute lymphoblastic leukemic cell line HSB-2 was kindly provided by Dr F. Preyers (Academic Hospital UMC Radboud, Nijmegen, The Netherlands). The human promyelocytic leukemic HL60 cell line and the human acute myeloid leukemic Kasumi-1 cell line were obtained from the German Collection of Micro-organisms (Braunschweig, Germany). Cells were cultured in RPMI-1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (RPMI⁺ medium). Supplements and antibiotics were obtained from Life Technologies (Grand Island, NY). Cell cultures were maintained in a 5% CO₂ humidified atmosphere at 37°C. The medium was refreshed every 3–4 days. For the experiments, exponentially growing cells were used. Tissue culture disposables were supplied by Corning (Badhoevedorp, The Netherlands).

Apoptosis inducing treatments

Apoptosis was induced by heat using a thermostatically controlled water-bath filled with distilled water to heat the cell suspension at 42.5°C for 1 h. Tissue culture flasks containing 10 mL of cell suspension (0.5 × 10⁶ cells/mL) were submerged into the water-bath. Temperature was monitored in all experiments with a flask containing only medium and a temperature probe to ensure a temperature of 42.5±0.2°C. For irradiation experiments, cells were exposed to 6 MeV X-rays at a rate of 5 Gy/min, to a total dose of 8 Gy with a Clinac600 6 MV linear accelerator (Varian, Silicon Valley, USA). In order to inhibit caspase-2 activity, the caspase-2 specific inhibitor zVDVAD-fmk (R&D systems, Minneapolis, USA) was added to the cell suspension 1 h before the experiments at a final concentration of 10 µM.

Quantification of Bcl-2 and Bax protein levels

Intracellular Bcl-2 and Bax protein levels were determined by flow cytometry 2 h after induction of apoptosis. Cells (0.3 × 10⁶) were fixed and permeabilized using an Intraprep kit (Immunotech, Marseille, France) and subsequently incubated with 1 µg anti-Bcl-2-FITC (IgG1, Dako, Glostrup, Denmark) or 1 µg anti-Bax-PE (IgG1, Brunschwig, Amsterdam, The Netherlands). Controls were performed by incubating cells with 1 µg mouse IgG1-FITC or IgG1-PE to measure non-specific fluorescent signals. Incubations with antibodies were all performed at room temperature for 20 min. Cells were then washed twice with Isoton and resuspended in 500 µL PBS. The mean fluorescent ratio (MFR), defined as the ratio of the mean fluorescent intensity (MFI) of the primary antibody and the control isotype stained cells, was used to measure Bcl-2 or Bax expression.

Monitoring cytochrome c release

Cytochrome c release from mitochondria to the cytosol was monitored using flow cytometry based on the technique reported by Campos et al. [17]. In this technique the plasma membrane of cells is selectively permeabilized by Digitonin or Saponin. The released cytochrome c is thereafter quickly washed out from the cells and what remains in the mitochondria is immunolabeled after fixing the cells. This enables the distinguishing between fractions of highly fluorescent cells that retained their mitochondrial cytochrome c and cells that lost their cytochrome c resulting in low fluorescence. Cells (0.5 × 10⁶) were permeabilized and fixed using an Intraprep kit (Immunotech, Marseille, France) with Saponin as permeability agent. Thereafter, cells were incubated with 5 µg anti-cytochrome c (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) at room temperature for 30 min. Subsequently, cells were washed twice and incubated with 1 µg polyclonal goat anti-mouse fluorescein isothiocyanite (FITC) conjugated secondary antibody (Dako, Glostrup, Denmark) at 4°C for 1 h. Finally, cells were washed twice, resuspended in 500 µL Isoton and immediately analysed by flow cytometry.

Measurement of apoptosis

Binding of Annexin-FITC (NeXins Research BV, Hoeven, The Netherlands) to PS as a measure of PS externalization and the uptake of propidium iodide (PI) (Sigma, St Louis, MO) as a measure for membrane integrity, were monitored by flow-cytometry [16]. Cells (0.3 × 10⁶) were washed twice with PBS and resuspended in freshly made HEPES buffer (Brunschwig, Amsterdam, The Netherlands) supplemented with 147 mM CaCl₂·5H₂O. Cells were incubated with 0.01 µg/mL Annexin V-FITC for 15 min at room temperature in the dark. Directly before measurements cells were stained with 0.2 µg/mL PI. Samples were kept on ice until flow-cytometric analysis and were measured within 1 h.

Flow cytometry

Fluorescence of individual cells was measured with a Coulter Epics XL flow cytometer. Excitation was elicited at 488 nm with the argon laser and emission was subsequently analysed by means of three fluorescence detectors; FL1 (530 nm band-pass filter), FL2 (585 nm band-pass filter) and FL3 (650 nm band-pass filter). In each sample 10,000 events were measured. Annexin V-FITC and PI were detected in FL1 and FL3 respectively. Cytochrome c indirectly labeled with GAM-FITC and FITC-labeled Bcl-2 were detected using the FL1 channel, while PE-labeled Bax was detected in FL2. Data are presented as percentage change in MFR of treated cells compared to MFR of untreated cells for Bcl-2 and Bax or as a percentage of cells with cytochrome c release compared to untreated cells.

Localization of Bcl-2 and Bax

Intracellular localization of Bcl-2 and Bax proteins was investigated by means of confocal microscopy 2 h after induction of apoptosis. Cells (10⁶) were washed twice in PBS, fixed for 30 min with 2% formaldehyde and incubated 25 min at 4°C. Subsequently, cells were washed in PBS and permeabilized for 5 min with 0.05% Triton X-100 in PBS. Non-specific antibody binding was blocked by incubation with 1% BSA in PBS for 1 h at room temperature. Anti-Bcl-2-FITC (IgG1, Dako, Glostrup, Denmark) or anti-Bax-PE (IgG1, Brunschwig, Amsterdam, The Netherlands) diluted in a 3% BSA/PBS to a final concentration of 1 µg/mL were added to the cells for 1 h at room temperature in the dark in a humidified atmosphere at 37°C. Excess antibody was removed by washing the cells four times with PBS. Thereafter, cells were spun down onto glass microscope slides with low acceleration (700 rpm for 10 min) using a cytospin3 micro-centrifuge (Shandon, Pittsburg, USA) and finally a coverslip was mounted onto the glass microscope slides using a mounting reagent (Dako, Glostrup, Denmark). Confocal laser scanning microscopy was performed with a Zeiss LSM 510-meta system using a 63× oil immersion objective. Excitation wavelengths and filters used were the following: FITC-stained Bcl-2, 488 nm excitation, emission BP 500-550 nm filter; PE-stained Bax, 543 nm excitation, LP 560 nm filter. A multi-track configuration was used for successive imaging of the two dyes. It should be noted that care was taken to decrease the laser intensity as much as possible to limit bleaching of the dyes and it was set on 5% and 15% for FITC-Bcl-2 and PE-Bax, respectively. The imaging parameters were 1024 × 1024 pixels format for the pictures and a 6.40 µs pixel time for sample scanning. In this way, high-resolution pictures with little fluorescent noise were obtained. For the semi-quantification of the fluorescence intensities measured for Bcl-2 and Bax, the intensity of green and red fluorescence was measured per cell by using ImageJ software. The total intensity values were corrected by the total surface area of the cell and compared to control cells in order to obtain the intensity per surface unit.

Statistical analysis

Data represent mean ± standard error of the mean (SEM) of three independent experiments. The effects of the different treatments (HT, RT, HT+RT and RT+HT) on Bax and Bcl-2 expression as well as cytochrome c release were analysed by 1-way repeated-measures ANOVA. After the between-groups analyses the post-hoc Student's t-test was performed to test for the significance between treated and untreated cells. Differences in relative Bax increase and cytochrome c release in the presence of zVDVAD-fmk between experimental groups were analysed using an unpaired two-tailed Student's t-test. Results were considered significantly different at p values <0.05.

Results

Bcl-2 and Bax expression after heat treatment and X-irradiation

To assess the role of Bcl-2 and Bax in the regulation of mitochondrial outer membrane permeabilization, the expression of Bcl-2 and Bax was monitored after heat treatment and irradiation. The hematopoietic cell lines HL60, HSB2 and Kasumi-1 were exposed to heat and/or irradiation and samples were obtained 2 h after treatment. Heat treatment induced clear quantitative changes in Bax protein expression compared to control cells as measured with the flow cytometer (Figure 1a). The increase in Bax expression after heat treatment of the three cell lines was dependent on the cell type ranging from 93% increase for HL60 cells to 228% for the more heat sensitive Kasumi-1 cells. In contrast to the increase of Bax expression in response to a transient increase of temperature, Bax expression after X-irradiation showed no increase in HL60 cells and a slight increase in HSB2 and Kasumi-1 cells.

Figure 1. Intracellular Bcl-2 and Bax protein levels. (a) Percentage increase in Bax MFR 2 h after treatment compared to Bax MFR of untreated cells. (b) Percentage decrease in Bcl-2 MFR 2 h after treatment compared to untreated cells. The experiments were performed on three cell lines HL60, HSB2 and Kasumi-1. The treatments given were heat treatment (HT), irradiation (RT), heat treatment directly followed by irradiation (HT+RT) and irradiation directly followed by heat treatment (RT+HT) (See also Materials and methods, Apoptosis inducing treatments). The MFR, defined as the ratio of the mean fluorescent intensity (MFI) of primary antibody and the MFI of the isotype control stained cells, was used as a measure for Bcl-2 or Bax protein expression. The results are the mean ± SEM of three independent experiments. †Indicates a p < 0.01 between the groups RT and HT, RT and HT+RT, RT and RT+HT as determined by 1-way ANOVA. *p < 0.05, **p < 0.01 relative to control cells as determined by Student's t-test.

Another aspect we were interested in was whether the sequence of treatments in the multimodal application of heat treatment and irradiation had an effect on the Bax and Bcl-2 expression levels. When heat and X-ray treatments were combined, an additive effect was observed for Bax expression and this was independent of the order in which heat treatment and irradiation were applied. The Bax increase after heat treatment alone or in combination with RT was significantly different in all three cell lines compared to cells treated with X-irradiation alone (p < 0.01, 1-way ANOVA). In contrast to the significantly (p < 0.05) increased Bax expression levels compared to untreated cells, the decrease of Bcl-2 expression levels after heat- and/or radiation treatment was constant and independent of the treatment and order of treatments (Figure 1b). In addition, the decrease of Bcl-2 protein expression due to heat treatment or irradiation was independent of the cell type used.

Intracellular localization of Bcl-2 and Bax

A possible co-localization of Bcl-2 and Bax after heat treatment or X-irradiation was investigated by confocal microscopy. In untreated HL60, HSB2 and Kasumi-1 cells Bcl-2 was localized in the cytoplasm (Figure 2a, d and g). In contrast to HL60 and HSB2 cells, untreated Kasumi-1 cells expressed very little Bcl-2. Bax was little or not expressed in untreated cells or even absent. Two h after heat treatment, Bax expression homogeneously increased 11.1, 6.4 and 3.0 fold in HL60, HSB2 and Kasumi-1 cells, respectively (Figure 2b, e and h). Bcl-2 condensed in the cytoplasm of all three cell types as a result of cell shrinkage due to apoptosis. However, this condensation did not result in the increase of Bcl-2 intensity per surface unit. Furthermore, Bcl-2 and Bax co-localized after heat treatment as observed in the overlay pictures (Figure 2b, e and h). All three cell types showed a small increase in Bax expression after X-irradiation of 1.6, 4.0 and 4.1 fold for HL60, HSB2 and Kasumi-1 cells respectively, as compared to untreated cells. Furthermore, Bax was homogeneously dispersed in the cytoplasm and did not co-localize with Bcl-2 after X-irradiation (Figure 2c, f and i).

Figure 2. Localization and expression of Bcl-2 and Bax. Images of Bcl-2 (green fluorescent) and Bax (red fluorescent) proteins in HL60, HSB2 and Kasumi-1 cells. Samples were taken of untreated control cells and 2 h after heat treatment (HT) or irradiation (RT). The cells were fixed and stained as described in Materials and methods. Each confocal microscope image (a-i) is built up by 4 pictures namely Bcl-2 alone, bright field, Bax alone and overlay of Bcl-2 and Bax (from left to right, top to bottom). Bax and Bcl-2 fluorescence intensities were quantified using ImageJ software corrected for the surface area and compared to the fluorescence intensities of control cells, see results. Images are representative of three independent experiments.

Release of cytochrome c into cytosol

The release of cytochrome c through the outer mitochondrial membrane into the cytosol was monitored by flow cytometry 6 h after heat treatment or X-irradiation. In control samples a maximum of 10% of the cells (within the total cell-population of 10,000 cells) showed cytochrome c release into their cytosol. Heat treatment of HL60 and HSB2 cells resulted in the release of cytochrome c in 50-60% of the cells, whereas approximately 90% of the Kasumi-1 cells released cytochrome c from the mitochondria (Figure 3). This increased release of cytochrome c was significantly different for all three cell types (p < 0.01), as compared to untreated cells. The difference in cytochrome c release correlates directly with differences in Bax expression after heat treatment as shown in Figure 1a. Similar observations were made for X-ray treatment that led to a small increase of Bax expression and consequently a relatively low but significant (p < 0.05) increase in the percentage of all three cell types releasing cytochrome c. The cytochrome c release after HT was significantly different from the cytochrome c release after RT in all three cell lines (p < 0.05, 1-way ANOVA).

Figure 3. Cytochrome c release into the cytosol. Percentage of cytochrome c release 6 h after heat treatment (HT) or irradiation (RT) compared to untreated cells (For details see Materials and methods, Apoptosis inducing treatments). The results are the mean ± SEM of three independent experiments. †Indicates a p < 0.05 between the groups HT, RT and control cells as determined by 1-way ANOVA. *p < 0.05, **p < 0.01 relative to control cells as determined by Student's t-test.

Effect of zVDVAD-fmk on Bax expression, cytochrome c release and apoptosis

In order to investigate the role of caspase-2 in the initiation of heat- and radiation-induced apoptosis, Bax expression levels and cytochrome c release were monitored after heat treatment and X-irradiation in the presence of the specific caspase-2 selective inhibitor zVDVAD-fmk. Blocking caspase-2 activation by adding zVDVAD-fmk 1 h prior to treatments resulted for all three cell types in a significant inhibition of Bax expression after heat treatment (Figure 4a). In contrast, Bax expression levels remained the same after irradiation of cells in the presence of the inhibitor.

Figure 4. Effect of caspase-2 inhibitor on Bax expression and cytochrome c release. (a) Percentage increase in Bax MFR 2 h after treatment compared to Bax MFR of untreated cells. The treatments given were heat treatment (HT) and irradiation (RT) (for details see Materials and methods, Apoptosis inducing treatments). The HL60, HSB2 and Kasumi-1 cells were incubated with (+) or without (−) 10 μM zVDVAD-fmk for 1 h before treatment. The results are the mean ± SEM of three independent experiments. * indicates a significant difference in relative increase in Bax expression compared to heat-treated cells without zVDVAD-fmk (p < 0.05, as determined by Student's t-test). (b) Percentage of cells with cytochrome c release 6 h after heat treatment (HT) or irradiation (RT) compared to untreated cells. The HL60, HSB2 and Kasumi-1 cells were incubated with (+) or without (−)10 μM zVDVAD-fmk for 1 h before treatment. The results are the mean ± SEM of three independent experiments. **Indicates a significant difference in cytochrome c release compared to heat treated cells without the presence of zVDVAD-fmk (p < 0.05, as determined by Student's t-test).

There was a significant decrease in cytochrome c release after heat treatment to nearly zero in all cells in the presence of zVDVAD-fmk (Figure 4b). In addition, zVDVAD-fmk treatment resulted in a decrease from 22% to 10% in the apoptotic population of HL-60 cells measured by Annexin V binding 24 hrs after heat treatment (data not shown). On the other hand, zVDVAD-fmk had no effect on radiation-induced cytochrome c release and this was in agreement with the unaltered intracellular Bax levels in the presence of the inhibitor.

Discussion

Apoptosis induced by mild hyperthermia proceeds via the mitochondrial pathway. A key step in the mitochondrial pathway of apoptosis is the activation and subsequent interaction of the multidomain BH3 proteins Bax and Bcl-2 in order to permeabilize the mitochondrial outer membrane as indicated in several studies [18–21]. In our previous study we showed that the commitment to apoptosis of HL60 promyelocytic cells after heat treatment starts with the mitochondrial membrane transition and is mainly executed in a caspase-dependent way [16]. To further elucidate the mechanisms involved in the induction of apoptosis by hyperthermia we monitored Bax and Bcl-2 expression levels after heat treatment in three hematopoietic cell lines. Furthermore we investigated the possible role of caspase-2 to activate Bax by inhibiting caspase-2 activity before the treatment of cells.

We observed that the mitochondrial outer membrane permeabilization is mediated by Bax expression resulting in the release of cytochrome c. Bax, and not Bcl-2, protein levels were affected fast after exposing cells to a transient increase of temperature. This increase in Bax expression shifts the balance between pro- and anti-apoptotic Bcl-2 family members towards apoptosis and triggers the release of cytochrome c most probably by the ability of Bax to form pores in the outer mitochondrial membrane [22], [23]. This correlation between Bax accumulation and cytochrome c release was also observed in our study showing that more cytochrome c is released into the cytosol in cells which accumulated more Bax after heat treatment.

When blocking activated caspase-2 by zVDVAD-fmk, Bax expression significantly inhibited MOMP, as indicated by significantly less release of cytochrome c and 50% inhibition of the Annexin V binding as an indication for apoptotic cell death. Our observations are in good agreement with the current view that caspase-2 acts on the mitochondria by regulating Bax via the BH3-only protein Bid, indicating that caspase-2 is essential for the cleavage of Bid into tBid. Furthermore, this model is supported by the results of Green et al., in which Bid-deficient mouse embryonic fibroblasts do not show any release of cytochrome c into the cytosol after heat treatment [9]. The present study is the first report to our knowledge in which the role of caspase-2 in heat-induced apoptosis is assessed by specifically inhibiting caspase-2 activation and shows that this caspase has a role upstream of the mitochondrial pathway. In contrast, another study in this field does not present any evidence in the depletion of caspase-2 and the role of any other initiator caspases on heat-shock induced apoptosis [24]. This led to the idea that initiator caspases might play a redundant role in promoting heat-induced apoptosis. However, it must be stressed that these experiments were carried out with a more intense heat shock (45°C over 2 h) which is fundamentally different from the mild hyperthermia used in the present study (42.5°C over 1 h).

Although an increase in Bax expression levels after heat treatment was detected in all three cell lines suggesting a general phenomenon, the amount of Bax increase was dependent on the cell type. Cell type characteristics, such as the presence of wild type tumor suppressor p53 protein, determine the regulation of MOMP by inhibiting Bcl-2 or activating/up-regulating Bax. As illustrated by the FCM data presented in this study, the wild type p53 expressing cell line Kasumi-1 showed significantly higher Bax expression levels after heat treatment compared to the HSB2 and HL60 cell lines which are depleted of functional p53. In addition, heat treatment affects not only the expression levels of Bax, but is also well known to modulate the expression levels of heat shock proteins (Hsp) via activation of the transcription factor heat-shock factor 1 (HSF-1) [25]. A member of these Hsp family proteins namely Hsp70, together with its co-chaperone Hsp40, is known to prevent Bax translocation into the mitochondria [26]. In a different study we investigated the role of Hsp70 on the apoptotic response after heat treatment and found that increased levels of Hsp70 inhibited apoptosis, and in a cell-type dependent way. These are only a few examples of interactions and feedback loops common in a complex pathway such as apoptosis, showing that Bax-mediated permeabilization of the mitochondrial outer membrane is influenced by numerous endogenous factors and is subsequently cell-type dependent.

It is proven that in the absence of Bax MOMP does not occur [27], stressing thereby the essential role of this protein in the intrinsic apoptotic pathway. Moreover, the anti-apoptotic Bcl-2 can prevent MOMP. The precise mechanism involved is controversial and several models have been proposed in which Bcl-2 and Bax interact with each other via their BH3 domains [28]. Therefore, heterodimeric interactions of Bcl-2 with pro-apoptotic Bax most probably occur in order to initiate MOMP. In this study we have shown that in the case of heat treatment, and not irradiation, Bcl-2 and Bax co-localize in the cytoplasm reinforcing the hypothesis of heterodimerization of these proteins. Furthermore, confocal images in agreement with FCM measurements (data not shown) showed differences in Bcl-2 expression for untreated cells whereby the HL60 and HSB2 cells showed higher expression levels of Bcl-2 compared to Kasumi-1 cells. Elevated Bcl-2 levels have been found in many types of cancers and promote resistance to chemo- and radiotherapy [29] and most probably also to hyperthermia. This is in agreement with the finding that cell lines with a high basal Bcl-2 expression level (HSB2 and HL60) showed less cytochrome c release as compared to the low Bcl-2 expressing cell line Kasumi-1 after heat treatment.

Hyperthermia is normally applied in the clinical setting together with other cancer treatment modalities (multimodal oncological strategies) such as radiotherapy in order to obtain an additive or synergistic interaction in vivo [30], [31]. For the combination of radiotherapy and hyperthermia the highest synergistic effects were shown in literature by simultaneous application [32], but this is not feasible in clinical practice. Therefore, the sequence of treatments is very important. However, in this in vitro study the combination of heat treatment followed by irradiation or vice-versa resulted in an additive effect with respect to Bax, but not Bcl-2 expression. Based on our results no clear differences in Bax or Bcl-2 expression levels could be detected when changing the sequence of treatments. The known synergistic effect described in literature when hyperthermia and radiotherapy are combined is most probably not achieved in the onset of apoptosis by the regulation of MOMP by Bax or Bcl-2, but more likely downstream of mitochondria with the interaction of e.g. heat-shock proteins with important players of the apoptotic pathway such as Apaf-1 [33].

In conclusion, as a response to mild heat treatment, such as used in hyperthermia in clinical settings, the intrinsic mitochondrial pathway of apoptosis is triggered. The expression of pro-apoptotic Bax is affected by heat treatment and plays a critical role in mediating the MOMP and the release of cytochrome c. Caspase-2 is crucial for heat-induced apoptosis as indicated by the inhibition of both Bax expression and cytochrome c release after blocking the caspase-2 activity. The results presented in our study are in good agreement with the current view that cytotoxic stress causes activation of caspase-2, and that this caspase is required for the permeabilization of the outer mitochondrial membrane. These understandings broaden our knowledge about hyperthermia and this is needed to increase the anti-tumor effect of hyperthermia alone or in multimodal strategies with radiotherapy.

Acknowledgements

This work was financially supported by the University of Twente as part of the strategic research project NIMTIK (Non-Invasive Molecular Tumor Imaging and Killing).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.