Combination of hyperthermia and bortezomib results in additive killing in mantle cell lymphoma cells

Abstract

The proteasome inhibitor bortezomib exhibits antitumor activity in many malignancies including mantle cell lymphoma (MCL). Unfortunately, many patients fail to respond to treatment or become refractory. Hyperthermia is an effective chemosensitizer that in combination with some chemotherapeutic agents has shown clinical activity in phase II and III studies. The aim of this study was to use MCL cell lines to investigate the potential benefit of combining clinically relevant doses of bortezomib with two different thermal doses (41.8°C/120 min and 44°C/30 min) that mimic the heterogeneity of the temperature distributions achieved within tumors during hyperthermia. Treated tumor cells were assessed for proliferation using the WST-1 assay and for apoptosis by annexin V staining, while heat shock protein (HSP) levels were determined following western blot analysis. Our results demonstrated that MCL cell lines that are sensitive to bortezomib are also thermosensitive and have low basal expression of hsp27, whereas the bortezomib-resistant MCL cell line strongly expresses hsp27 and is thermoresistant. Interestingly, pre-treatment of MCL cell lines with heat at the two different thermal doses, and the transient elevation of hsp27 and hsp70, do not impair their primary sensitivity to bortezomib. Finally, we show that the concurrent treatment of heat and bortezomib results in additive killing in MCL cell lines.In conclusion, these results suggest that the application of bortezomib, under thermal conditions, in mantle cell lymphoma cells may be beneficial and warrants further investigation.

• Heat shock proteins
• hyperthermia
• proteasome inhibition
• mantle cell lymphoma

Introduction

Bortezomib (Velcade®) is a potent, selective, and reversible inhibitor of the 26S proteasome. The proteasome is composed of a 20S core particle where proteolysis takes place and a 19S thermosensitive regulatory particle. Substrates include various oncogenes and regulatory proteins for cell cycle progression and apoptosis 1. The ability of proteasome inhibitors to disrupt cell cycle progression has made this class of small molecule inhibitors attractive chemotherapeutic agents 2,3. Bortezomib demonstrated clinical activity in multiple myeloma, for which it gained FDA and EMEA approval in 2004, and in many other malignancies 3–5 including mantle cell lymphoma 6,7. Mantle cell lymphoma is a unique subtype of B-cell malignant lymphoma that behaves aggressively and remains incurable 8,9. Although recently, phase II studies showed that bortezomib as a single agent can achieve durable and complete responses in MCL, many patients fail to respond to treatment or become refractory after an initial response 6,10,11. Therefore, efforts have been made to understand the mechanisms responsible for resistance and sensitivity to bortezomib, and to develop strategies that can overcome this resistance.

Hyperthermia is a cancer therapy that has been shown to have clinical activity when combined with radiation or chemotherapy 12–14. This improved response to radiation or drugs can result from a simple additive effect due to the heat treatment inducing a direct cytotoxicity effect in the tumor cells, or by sensitization in a temperature-dependent manner 15. It is known that heat causes a greater than additive cell killing when combined with alkylating agents, nitrosureas, and platinum drugs. In the case of cisplatinum, heat has been found to reverse cisplatinum resistance 16,17, leading to a more general suggestion that hyperthermia could be instrumental in reversing drug-resistance. Thus, heat may be an ideal candidate for combination with bortezomib.

Heat shock proteins are molecular chaperones involved in the regulation of various vital cellular functions, including cell proliferation 18. Although HSPs are only induced transiently in cells after periods of stress as can occur with heat, they are often constitutively overexpressed in tumor cells 19. Indeed, HSPs are commonly expressed in MCL 20,21 and in particular hsp90 overexpression has been found to correlate with the high-grade type of lymphoma 21. Recently, overexpression of heat shock proteins such as hsp27, hsp70 and hsp90 has been found to be associated with bortezomib resistance in B-lymphoma cells 22. A major concern, therefore, is the possible induction or increase of bortezomib resistance by heat-induced overexpression of HSPs.

In the present study, we aimed to evaluate the time- and temperature-dependent effects of heat exposure in three lymphoma cell lines that displayed differential sensitivity to bortezomib. Based on our kinetic studies, the time of bortezomib exposure for combination experiments was set to 24 h, a window where bortezomib shows an anticancer activity in all mantle cell lymphoma cell lines but does not kill the majority of the cells, allowing study of potential synergistic or additive effect of other anticancer agents or modalities. We investigated the combination of heat and bortezomib comparing two thermal doses, a severe and a mild thermal dose that mimic the heterogeneity of the temperature distribution achieved within tumors during hyperthermia treatment, and two different time points of the heat shock response which correspond to the basal and maximal expression of HSPs, to explore their role in influencing bortezomib sensitivity.

Materials and methods

Tumor cell lines and reagents

Seven cell lines were used. These included five tumor cell lines: Jeko-1, Rec-1, Granta 519, HBL-2 and NCEB-1, and two haematological control cell lines: Jurkat (T-ALL) and Karpas 422 (diffuse large cell lymphoma). All cell lines were either purchased from the German Collection of Microorganisms and Cell Lines (Deutsche Sammlung von Mikroorganismen und Zelllinien, Braunschweig, Germany) or provided by the principle investigators. Jeko-1, Granta 519, and NCEB-1 are human mantle cell lymphoma cell lines established from three different patients, a 78-year-old woman with B-cell non-Hodgkin's lymphoma (NHL), a 58-year-old woman with B-cell NHL, and a 57-year-old male with diffuse centroblastic-centrocytic B-NHL, respectively. Rec-1 and HBL-2 were established from the lymph nodes of two men who had terminal refractory B-cell non-Hodgkin lymphoma and were 61 and 74 years old, respectively. All cells were incubated at 37°C in a humidified atmosphere at 5% CO₂/95% air and maintained in RPMI 1640 medium with L-glutamin plus 2 g/L NaHCO₃ supplemented with 20% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. The proteasome inhibitor bortezomib (PS-341) was provided by Millennium Pharmaceuticals (Cambridge, MA) aliquoted and stored at −20°C.

Bortezomib treatment

Fixed cell numbers were incubated in the presence of increasing concentrations of bortezomib and harvested at several time points (12, 24, 48 and 72 h) and assayed for viability, survival, and apoptosis. For the combination experiments, escalating concentrations of bortezomib (ranging from 3.125 nM to 100 nM) were added shortly before or 24 h after heating at 41.8°C for 120 min or 44°C for 30 min in a temperature-controlled water bath 23. Control cells were incubated at 37°C with the same drug concentrations for exactly the same time. The inhibitory concentration of bortezomib was expressed as the IC₅₀ dose (the half maximal inhibitory concentration), which represents the drug concentration that is required to inhibit 50% of the target cells.

Heat treatments

Heat exposure as a function of time was performed using two thermal doses: 41.8°C for 120 min and 44°C for 30 min, which mimic the heterogeneity achieved during clinical heat treatment. Heating was performed by directly immersing the cell culture flasks, sealed with parafilm, in a temperature-controlled water bath 24. Control cells were sealed and incubated at 37°C for exactly the same time as the heat-exposed samples. After treatment, the cells were returned to 37°C/5% CO₂ and allowed to recover for up to 24 or 48 h. At these defined recovery time points, non-viable cells in the supernatant were removed and viable cells were harvested for analysis.

Cell viability, apoptosis, and cell proliferation assays

Cell viability was evaluated at several time points after treatment using the Trypan blue dye-exclusion assay. Only viable cells were harvested and treated with bortezomib. For the apoptosis assay cells were washed and stained with Annexin V-PE/7-ADD in accordance with the manufacturer's protocol (BD Biosciences, Heidelberg, Germany) and subjected to flow cytometry. The degree of apoptosis was calculated from the formula:

For the established MCL cell lines the spontaneous apoptosis rate was 10%. Cell proliferation was measured using the WST-1 Cell Proliferation Assay (Roche Diagnostics, Mannheim, Germany). Briefly, lymphoma cells were exposed to 41.8°C/120 min and 44°C/30 min, and at defined time-points viable cells were harvested, seeded and incubated in 96-well culture plates in the presence of increasing concentrations of bortezomib for 24 h at 37°C. WST was then added to each well for 4 h at 37°C. Absorbance was measured at 450 nm/690 nm using a standard reader (Optimax Pro, Molecular Devices; Sunnyvale, CA). Cell proliferation was estimated as a percentage of the untreated control cells (37°C). Triplicate wells were used for each experimental condition and all experiments were repeated at least three times.

Western blot analysis

Mantle cell lymphoma cells were heated at 41.8°/120 min or 44°C/30 min and allowed to recover at 37°C for different times (0 to 48 h). An equal amount of viable cells were harvested, washed, and lysed in 100 µL buffer (150 mM NaCl; 50 mM TRIS, pH 8; 1% NP40) containing 1 µL/mL leupeptin and 2 µL/mL of protease inhibitors. Cells were incubated for 30 min on ice, centrifuged for 15 min at 4°C and boiled for 10 min at 95°C.

Cell lysates were loaded onto one lane and separated by SDS-PAGE. Cell lysates adjusted for an equal number of cells and an equal quantity of proteins (measured by the Bradford assay) were compared. After electrophoresis, proteins were transferred to nitrocellulose membranes (Sartorius Nitrocellulose Blotting Membranes, Göttingen, Germany) and probed overnight at 4°C with appropriate antibodies. The following monoclonal antibodies were used: the rat antibody IgG1 6B3 25 against hsp70, a mouse antibody against hsp27 (SPA-800, Stressgen Bioreagents), a mouse antibody against hsp90 (SPA-830, Stressgen), and a rat antibody against Hsc70 (SPA-815, Stressgen). A secondary peroxidase-conjugated mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, PA) or goat anti-mouse IgG (Bio-Rad Laboratories, CA) (1:2000) was added for 1 h at room temperature. Detection of the protein/antibody complexes was achieved using the ECL system (Amersham Life Science, Karlsruhe, Germany).

Statistical analysis

The statistical significance of experimental values was assessed by means of an independent Student's t-test on two populations. P-values of p < 0.05 were considered significant.

Results

Time- and dose-dependent differential sensitivity of mantle cell lymphoma cell lines to bortezomib

The kinetics of cytotoxicity, the degree of apoptosis, and antiproliferative activity induced by treatment with bortezomib was evaluated in all cell lines. For assessing viability, cells were incubated with increasing concentrations of bortezomib (0 nmol/L to 50 nmol/L) and harvested at 24, 48 and 72 h. In all cell lines a dose- and time-dependent inhibition of cell growth was observed, with differential sensitivity to the proteasome inhibition among the cell lines. These effects are illustrated in Table I and Figure 1. Table I (left panel) shows the time dependency using a clinically achievable concentration of bortezomib (25 nM) 23 and demonstrates a strong inhibition of cell growth in Jeko-1, HBL-2 and Karpas 422 within 48 h, whereas in NCEB-1 the same exposure time only inhibited growth by 30%. Figure 1 shows the drug concentration dependency after 24 h of treatment in three of the mantle cell lymphoma cell lines. From these results the cell lines can be ranked as having high (Jeko-1), intermediate (Rec-1), or low (NCEB-1) sensitivity to bortezomib.

Figure 1. Cell survival expressed as percentage of proliferating cells of Jeko-1, Rec-1 and NCEB-1 in the presence of increasing concentrations of bortezomib. Cells were incubated with bortezomib (from 0 to 100 nmol/L) for 24 h in a humidified atmosphere at 37°C. Cells were harvested and the relative number of proliferating cells after treatment was assessed by standard WST-1 assay. Error bars represent mean ± standard deviation of triplicates in one representative experiment.

To detect the early and late apoptosis events induced by bortezomib at the clinically relevant concentration (25 nM), viable cells were analysed for apoptosis using flow cytometry after 12 and 24 h of drug exposure. NCEB-1 again arose as the most resistant cell line and Jeko-1 the most sensitive, whereas Rec-1, HBL-2 and Granta 519 showed an intermediate sensitivity (data not shown).

Table I.  Kinetics of viability in MCL and haematological control cell lines following bortezomib treatment (left column). Cells were incubated with 25 nM of bortezomib, harvested at 24, 48 and 72 h and assessed for viability. The right column lists the IC₅₀ values derived from the proliferating activity assessed by WST-1 assay in all cell lines treated for 24 h with increasing concentrations of bortezomib.

Cell lines^1	Bortezomib concentration	0 h	24 h	48 h	72 h	IC50² (nmol/L)
Jeko-1	0 nM	100	186	286	449
	25 nM	100	57	6	0	14.7 ± 2
Rec-1	0 nM	100	149	167	298
	25 nM	100	55	28	6	25.6 ± 2
NCEB-1	0 nM	100	126	184	298
	25 nM	100	78	71	6	33.8 ± 7
Granta 519	0 nM	100	146	200	273
	25 nM	100	47	13	3	25.2 ± 4.2
HBL-2	0 nM	100	189	270	348
	25 nM	100	42	5	0	22.4 ± 2.6
Jurkat	0 nM	100	146	217	294
	25 nM	100	76	43	26	24.4 ± 0.3
Karpas 422	0 nM	100	226	302	391
	25 nM	100	48	4	1	20.3 ± 2.2

¹Values are the mean of three independent experiments. Viability is expressed as percentage of untreated control cells set to 100%.

²IC₅₀ values after 24 h treatment with increasing concentration of bortezomib.

Finally, the antiproliferative activity of bortezomib at increasing concentrations (3.125 nmol/L to 100 nmol/L) for 24 h in the various cell lines were compared by determining the IC₅₀ doses, and confirmed the previous pattern of sensitivity. The highest IC₅₀ value was again achieved with NCEB-1. This was followed by Rec-1, Granta 519, Jurkat, HBL-2, Karpas 422 and finally Jeko-1 (Table I, right panel).

In conclusion, cytotoxicity, antiproliferative activity and apoptosis assays consistently showed that exposure to bortezomib differentially affects mantle cell lymphoma cells in a time- and dose-dependent manner. To explore the interaction and potential benefit of bortezomib in combination with hyperthermia, we selected three mantle cell lymphoma cell lines with high (Jeko-1), intermediate (Rec-1) and low sensitivity to bortezomib (NCEB-1) (Figure 1).

Thermal dose-related differential effects of heat on cell viability and survival of mantle cell lymphoma cells

Cells were exposed to a mild heat temperature of 41.8°C for 120 min and a more severe heat treatment of 44°C for 30 min. After heating, the cells were allowed to grow at 37°C/5% CO₂ for 24 h before being harvested. Viability and cell survival were assessed using the Trypan-blue exclusion and WST-1 assays, respectively. As expected, exposure of cells to the lower thermal dose (41.8°C/120 min) had no or minimal impact on survival and viability in all three cell lines selected. Survival rates expressed as a percentage relative to control cells treated at 37°C are illustrated in Table II (left panel). Exposure of Jeko-1 and Rec-1 to a more severe thermal dose (44°C/30 min) did reduce tumor cell survival, whereas for NCEB-1 cells no cell killing was observed after exposure to this larger thermal dose (Table II, right panel). These survival patterns show that heat differentially affects mantle cell lymphoma cells in a time- and temperature-dependent manner and suggest that NCEB-1 is more thermoresistant in comparison to Jeko-1 and Rec-1.

Table II.  Survival rate in Jeko-1, Rec-1 and NCEB-1 after heat treatment at a mild thermal dose (41.8°C/120 min, left column) and at a severe thermal dose (44°C/30 min, right column). Cells were first heated and allowed to recover at 37°C in a humidified athmosphere for 24 h. Proliferation rate was evaluated by a standard WST assay.

MCL^1	Control at 37°C^2	Heat exposure at 41.8°C/120 minutes	Heat exposure at 44°C/30 minutes
Jeko-1	100	94.5 ± 10.7	38.3 ± 19.7
Rec-1	100	73.9 ± 7.3	28.2 ± 10.5
NCEB-1	100	111.5 ± 11.6	100.9 ± 5.1

¹Rates are the mean of three independent experiments (±SD).

²Rates are the percentage of survival to control cells at 37°C, which was set to 100%.

Differential constitutive expression and heat-induced overexpression of heat shock proteins in mantle cell lymphoma cell lines

To compare the basal expression of HSPs in the three selected MCL cell lines at the physiological growth temperature of 37°C, an equal amount of viable cells were harvested and the expression level of the constitutively expressed Hsc70, hsp70, hsp27, and hsp90 were compared using western blot analysis. At 37°C, hsp70, hsp27, and hsp90 were detectable in all three cell lines, although the basal expression of the inducible hsp70 was low in all lines, and NCEB-1 expressed a much higher basal level of hsp27 than Rec-1 and Jeko-1 (Figure 2a).

Figure 2. Expression level of hsp27, hsp70, Hsc70, and hsp90 by western blot analysis in an equal number of Jeko-1, Rec-1, and NCEB-1 cells at the physiological growth temperature of 37°C (a). Kinetics of expression of hsp27, hsp70, Hsc70, and hsp90 in Jeko-1, Rec-1 and NCEB-1 after exposure to a mild thermal dose (41.8°C/120 min) (b), and to 44°C/30 min (c). ß-actin was used as a protein loading control. After heat treatment at the selected thermal doses cells were allowed to recover in a humidified atmosphere at 37°C for up to 48 h. At the time points 4, 16, 24 and 48 h an equal numbers of cells were harvested for western blot analysis. Control cells were kept at 37°C (left column of each panel).

The characteristics of the induction of the heat shock proteins following heating in the selected lymphoma cell lines was assessed by treating cells with the two thermal doses (41.8°C/120 min and 44°C/30 min) and allowing the cells to recover at 37°C for 4, 16, 24, and 48 h. At these time points the expression levels of Hsc70 (constitutive), hsp70 (inducible), hsp27, and hsp90 were investigated using western blot analysis by comparing both an equal number of cells and an equal quantity of proteins among the cell lines (data not shown). Figures 2b and 2c show the kinetics of HSPs expression in Jeko-1, Rec-1 and NCEB-1.

The basal expression of the constitutive form of the hsp70 family (Hsc70) and of hsp90 in all three cell lines, as expected, was not impaired by heat exposure at both thermal doses. Inducible hsp70 was transiently overexpressed with a peak between 16–24 h of recovery after exposure to heat treatment at 41.8°C/120 min (Figure 2b) and 44°C/30 min (Figure 2c) in all three cell lines, whereas hsp27 was strongly induced only in Rec-1 and Jeko-1 but not in NCEB-1 (Figures 2c and 2b).

The degree of increase of inducible HSPs is thermal dose-dependent as demonstrated by the stronger increase of hsp70 and hsp27 after exposure to 44°C/30 min compared to 41.8°C/120 min (Figures 2b and 2c).

Pre-treatment with heat and heat-induced overexpression of heat shock proteins does not interfere with the effects of bortezomib in MCL cell lines regardless of the thermal dose

To investigate whether or not the upregulation of HSPs by heat shock treatment correlates with an increase or induction of bortezomib resistance, the three selected MCL cell lines with differential sensitivity to bortezomib and heat were first treated at 41.8°C/120 min or 44°C/30 min and then allowed to grow at 37°C for 24 h. After this time of recovery viable cells were then incubated in the presence of increasing concentrations of bortezomib for an additional 24 h. The effect on cell survival of this combined treatment was assessed by WST assay. Figure 3 shows survival of Jeko-1, Rec-1 and NCEB-1 after treatment with bortezomib vs. the same treatment given 24 h of recovery after heat exposure to 41.8°C/120 min or 44°C/30 min. These survival curves showing the concentration dependency as well as the respective IC₅₀ values clearly show that the pattern of bortezomib sensitivity after heat treatment remained virtually unchanged in all three cell lines in comparison to bortezomib treated cells at 37°C regardless of the concentration of bortezomib or the thermal dose applied (Table III).

Figure 3. Cell survival expressed as percentage of proliferating cells in Jeko-1 (a), NCEB-1 (b), and Rec-1 (c), treated with increasing concentrations of bortezomib after a pre-treatment with heat at 41.8°C/120 min or 44°C/30 min. Briefly, cells were first heated, then allowed to recover for 24 h in a humidified atmosphere at 37°C. At this point cells were treated with bortezomib (from 0 to 100 nmol/L) for 24 h and then harvested for survival assessment by standard WST-1. Survival is expressed as a percentage of control cells (bortezomib-untreated) and compared with non-heated cells (37°C). Error bars represent mean ± standard deviation of three independent experiments. The percentage of proliferating cells at each concentration of bortezomib tested did not significantly differ between heated and non-heated cells and p-value was largely >0.5 (*).

Concurrent application of heat and bortezomib is additive in MCL cell lines

To investigate the biological effect of the combination of bortezomib and heat, mantle cell lymphoma cells were concurrently exposed to heat at the two selected thermal doses and increasing concentrations of bortezomib for 24 h. The effect on cell survival was assessed with the WST assay and compared using the IC₅₀ values, as summarized in Table III. In all three cell lines the IC₅₀ values, as well as the survival curves after bortezomib alone versus concurrent bortezomib and heat at 41.8°C/120 min or at 44°C/30 min (figure not shown) clearly demonstrate that there is no synergism regardless of the cell line, the thermal dose applied, or the bortezomib concentration used. In particular, in the most heat- and bortezomib-resistant NCEB-1 cell line, the combination treatment did not reverse resistance even at sub-therapeutic concentrations. However, when the survival rates of heated cells treated with bortezomib were compared to untreated control cells at 37°C, an additive effect was found in all three cell lines in particular at concentrations <25 nM, considered to be the clinically achieved concentration. Figure 4 shows the survival of cells expressed as percentage of proliferating cells treated at 44°C/30 min alone or with the combination of heat and bortezomib. At the IC₅₀ concentration, survival of heated cells in Jeko-1 and Rec-1 was about 20% compared to 50% in control cells (Figure 4, dotted lines), showing a clear additive effect of heat to the bortezomib treatment, whereas for NCEB-1 this additive effect was not evident at the IC₅₀ (Figure 4c).

Figure 4. Cell survival expressed as a percentage of proliferating cells in Jeko-1 (a), Rec-1 (b), and NCEB-1 (c), treated with increasing concentrations of bortezomib given at 37°C (black line) or given concurrently to heat at 44°C/30 min (grey line). Survival of heated cells is expressed as a percentage of untreated cells at 37°C. Briefly, cells were first treated with bortezomib (from 0 to 100 nmol/L) and immediately heated. After the concomitant treatment, cells were incubated for 24 h in a humidified atmosphere at 37°C. At this point cells were harvested for survival assessment by standard WST-1. Error bars represent mean ± standard deviation of four independent experiments. Dotted lines indicated the IC₅₀.

Overall, the combination of bortezomib and heat at a lethal temperature has an additive but not synergistic effect in thermosensitive MCL cell lines.

Table III.  IC₅₀ values obtained in Jeko-1, Rec-1, and NCEB-1 cells in the presence of increasing concentrations of bortezomib (from 0 to 100 nmol/L) for 24 h at: 37°C (control cells) listed in the left column; concomitant to heat treatment (middle column); or 24 h after pre-treatment with heat at the indicated thermal doses (right column).

	Bortezomib control	Bortezomib + heat at		Bortezomib + 24 hours after heat at
Cell lines	37°C	41.8°C/120 minutes	44°C/30 minutes	41.8°C/120 minutes	44°C/30 minutes
Jeko-1^1	14.7 ± 2	14.5 ± 1.7	13.9 ± 2.2	19.1 ± 3.5	11.8 ± 3.2
Rec-1	25.6 ± 2	30.7 ± 4.2	30.6 ± 10.6	33.7 ± 4.0*	34.2 ± 10.9*
NCEB-1	33.8 ± 6.9	34.2 ± 5.0	29.3 ± 16.6	40.7 ± 11.5	33.8 ± 9.0

*p > 0.05 in comparison to control group at 37°C.

¹Values (nmol/L) are the mean of three independent experiments (±SD). Only control values at 37°C of Jeko-1, Rec-1 and NCEB-1 were the mean of 13 and 11 independent experiments (±SD) respectively.

Discussion

Exposure to bortezomib affects mantle cell lymphoma (MCL) cells in a time- and dose-dependent manner, although variations occur among various cell lines (Table I and Figure 1). In the present study we evaluated the time- and temperature-dependent effects of heat exposure in combination with bortezomib in three mantle cell lymphoma cell lines (Jeko-1, Rec-1, and NCEB-1) that displayed high, intermediate and low sensitivity to bortezomib (Figure 1). We observed that cell lines sensitive to heat were also sensitive to bortezomib, whereas the most bortezomib-resistant cell line, NCEB-1, was also the most thermoresistant. All three MCL cell lines expressed basal levels of the heat shock proteins 70, 27, and 90 and in particular the most thermo- and bortezomib-resistant cell line NCEB-1 expressed a much higher basal level of hsp27 than the more sensitive cell lines (Figure 2a). It has been suggested that high basal expression of HSPs can protect against proteasome inhibitor-induced apoptosis; in particular Shringapure et al. showed a correlation between expression of hsp27, hsp70, and hsp90 and the bortezomib-resistant phenotype in a gene expression analysis of B-cell lymphoma 22. In our study in particular, we did not find a stronger basal expression of hsp70 or hsp90 but only of hsp27 in the more resistant cell line, supporting a previous observation that only hsp27 confers resistance to proteasome inhibition 22,26. Indeed, hsp27 blockade overcomes bortezomib resistance in lymphoma cells, whereas ectopic expression of wild-type hsp27 renders bortezomib-sensitive cells resistant 26.

It is generally assumed that HSP expression becomes deregulated in cancer, leading to elevated expression. Elevated HSP expression promotes cancer by inhibiting programmed cell death (hsp27, hsp70) and by promoting autonomous growth (hsp90), and that this leads to resistance to chemotherapy and hyperthermia 19. Many compounds have been recently developed that directly target the heat shock response by blocking HSF1 or HSP mRNA translation that dramatically sensitizes cancer cells to proteasome inhibitors 27. Inhibition of hsp90 by geldanamycin, moreover, has been found to exert an antilymphoma activity by increasing the proteasomal degradation of hsp90 client proteins, and to sensitize cancer cells to bortezomib 28,29. Consequently, a major concern in cancer therapy is the potential increase in drug resistance by the induction of HSPs.

In the present study we observed that tumor cells with much higher levels of hsp27 and hsp70 induced upon heat are no more resistant to bortezomib than tumor cells with low basal levels of these HSPs. Indeed, the maximal expression of hsp27 and hsp70 achieved between 16 and 24 h after heat exposure did not correlate with an increased resistance to bortezomib in Jeko-1, Rec-1, or NCEB-1 (Figure 3, Table III). This effect was found regardless of the thermal dose applied, indicating that the degree of inducible HSPs, which are higher after a more severe thermal stress, is not critical in conferring or impacting resistance. This observation is consistent with previous evidence that tumor cells with more HSPs are not more resistant to chemotherapy than those with low basal levels 30. Most importantly, it disproves the hypothesis that pre-treatment of cells with heat shock results in an increased resistance to proteasome inhibitors 27,31. A possible explanation for this is that, unlike thermotolerant cells in which transiently elevated levels of HSPs are freely available to interact with new substrates, the pool of HSPs in tumor cells could be engaged in chaperone complexes and unavailable for use in cytoprotection 19. In summary, we observed in our in vitro experiments that a pre-treatment with heat and the heat-induced overexpression of heat shock proteins 27 and 70 does not interfere with the sensitivity to proteasome inhibitors in MCL cell lines.

The response of cells to stress depends upon its severity, which is a product of intensity and duration. Also, the proteasome is a target of heat and impact of its activity is likely to correlate with the severity of heat. In agreement with previous findings 32,33 Pajonk et al. reported that severe heat shock at 44°C for 60 minutes rapidly inhibited the proteasome activity resulting in NF-κB inhibition, by the downregulation of the regulatory, thermosensitive subunit 19S in human prostate cancer cells 34. Conversely, it has been found that proteasome levels may even increase in response to long-term mild stress 1.

Hyperthermia modifies the cytotoxicity of many chemotherapeutic agents 15. The extent of thermal chemosensitization can be quantified by comparing clonogenic cell growth and proliferation activity in vitro or tumor growth inhibition in vivo when treated with the drug alone and with the same drug at elevated temperature. It is generally accepted that most alkylating agents (e.g., cyclophosphamide and ifosfamide), platinum compounds, and nitrosoureas (e.g., BCNU and CCNU) are linearly enhanced in their cytotoxic effect if temperatures are raised from 37°C to over 40°C, and exert more than an additive effect. In contrast, most antimetabolites (e.g., 5-fluorodeoxyuridin and methotrexate), as well as vinca-alkaloids or taxanes, show an independent action (additive effect). Studies on drug-heat sequence show, in general, that drugs administered immediately before or concomitantly to hyperthermia are most effective. The interaction between proteasome inhibitors and hyperthermia has been explored recently in a human colon cancer cell system 35. Chen et al. found a significant synergistic effect on cell viability with proteasome inhibition under hyperthermic conditions of 43°C for 60 minutes and showed that the concomitant combination of heat and proteasome inhibition decreased hsp27 levels, leading to thermal sensitization and increased apoptosis 35. The proposed mechanism of sensitization to both heat and to the proteasome inhibition through a decrease in hsp27 is plausible, as it is known that hsp27 blockade sensitizes both thermoresistant cells and cells resistant to chemotherapy in general and in particular to bortezomib. However, both heat shock as well as proteasome inhibition per se induce the heat shock response and a transient overexpression of inducible HSPs 2,36, so that this mechanism must be strongly time-dependent. In our experimental setting, the concurrent application of heat at two thermal doses and bortezomib in increasing concentration neither reversed bortezomib resistance in NCEB-1, that highly expressed high basal level of hsp27, nor produced more than additive killing (synergism) as a result of sensitization in Jeko-1 and Rec-1, regardless of the thermal dose applied. In our in vitro study the concomitant application of heat and bortezomib led merely to additive killing (independent action) at concentrations of bortezomib <20 nM and at thermal doses above the temperatures that induce exponential cell killing (Figure 4), and heat-induced overexpression of hsp27 did not interfere with bortezomib sensitivity. There are several possible explanations for the discrepancy in these results. Firstly, it has to be mentioned that NCEB-1 has five to eight stable murine chromosomes and expresses human and murine bcl-2 protein 37, so that possibly antiapoptotic genes may be differently regulated. Secondly, the MCL cell lines and colon cancer cells may indeed display different patterns of HSPs basal expression and behave differently following heat shock. Finally, the proteasome inhibitors were applied and their concentrations were different in the two systems; we chose increasing concentrations of bortezomib in a range (0–100 nM) that reproduces the clinically achievable concentrations 23, whereas Chen et al., used MG132 at much higher concentrations (10 µM), which are likely to result in different biological and pharmacological responses 35.

Overall, despite the intrinsic limitation of a merely in vitro study, our results suggest that the application of bortezomib under thermal conditions may be beneficial and deserves further clinical investigation. Moreover, our results suggest that MCL may be an appropriate tumor entity for this combinational treatment since MCL cell lines are sensitive to bortezomib at clinically relevant concentrations, can be killed by heat in a temperature-dependent manner, and the combined treatment results in at least additive killing.

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