Cell biological effects of hyperthermia alone or combined with radiation or drugs: A short introduction to newcomers in the field

Abstract

Hyperthermia results in protein unfolding that, if not properly chaperoned by Heat Shock Proteins (HSP), can lead to irreversible and toxic protein aggregates. Elevating HSP prior to heating makes cells thermotolerant. Hyperthermia also can enhance the sensitivity of cells to radiation and drugs. This sensitization to drugs or radiation is not directly related to altered HSP expression. However, altering HSP expression before heat and radiation or drug treatment will affect the extent of thermal sensitization because the HSP will attenuate the heat-induced protein damage that is responsible for radiation- or drug-sensitization. For thermal radiosensitization, nuclear protein damage is considered to be responsible for hyperthermic effects on DNA repair, in particular base excision repair. Hyperthermic drug sensitization can be seen for a number of anti-cancer drugs, especially of alkylating agents. Synergy between heat and drugs may arise from multiple events such as heat damage to ABC transporters (drug accumulation), intra-cellular drug detoxification pathways and repair of drug-induced DNA adducts. This may be why cells with acquired drug resistance (often multi-factorial) can be made responsive to drugs again by combining the drug treatment with heat.

• Protein denaturation
• protein aggregation
• radiosensitization
• chemosensitization
• heat shock proteins
• chaperone activity

Protein unfolding, heat shock protein expression and chaperone activity

Protein damage is the main molecular event underlying the biological effects of hyperthermia in the clinically relevant temperature range (39–45°C). The activation energies for protein denaturation and heat-induced cell death are within the same range [1]. Biophysical approaches, especially coming from the group of Lepock [2], [3], as well as work with model proteins [4], [5] have directly shown that substantial protein denaturation occurs in the clinically relevant temperature range. As a result of denaturation, proteins are prone to aggregation. Without chaperones, these aggregates can have destructive consequences for many macro-molecular structures and their function(s).

Despite the fact that it is known that protein damage plays a central role in the biological effects of hyperthermia, little is known about what finally kills the cells. Whereas heat-induced protein denaturation occurs randomly throughout the cell, nuclear proteins appear most sensitive [2], [3], [6] and/or the nuclear environment is the most favourable compartment for aggregation to occur [5], [7]. This is in agreement with the excellent correlations between nuclear protein aggregation and heat killing [8] and the extreme heat sensitivity of various nuclear processes [9]. Nevertheless, protein damage to other compartments and/or processes also could contribute to cell death after heat shock.

Expression of cell death after heat shock is cell type and heat dose dependent. Some cells (or a fraction of a cell population) may rapidly die due to apoptosis if the pathways to initiate and execute this programme are present in the cells and not inactivated (e.g. by high heat doses). Apoptosis defective cells will, nevertheless, not proliferate anymore, if the heat damage has been too extensive. They may lose their proliferative capacity via permanent cell cycle arrest (without disintegration of the cell), by necrosis or secondary apoptosis after S-phase or mitotic failure (Figure 1(a)).

Figure 1. Overview of some critical cell biological events induced by a hyperthermic treatment. (a) Schematical figure of the kinetics of HSP up-regulation after heating (closed squares), development of thermotolerance (TT) against a second heat treatment (closed circles), expression of cells death after the 1st heat treatment (closed diamonds), the magnitude of thermal radiosensitization expressed as thermal enhancement ratios (TER) being the ratio of doses required to kill a fixed percentage of cell with radiation alone or with heat plus radiation (closed triangles) and the amount of protein aggregation after heating (open squares) (see also (c)). (b) Schematical figure of magnitude of radiosensitization (expressed as TER) for a mild (triangles) or severe (diamonds) heat treatment given after (left), simultaneously with (grey area) or before (right) radiation. Cells made thermotolerant by prior heating (TT, squares) show reduced radiosensitization and more rapid loss of interaction for heat given before (but not after) radiation. (c) Radiosensitivity of cells when left unheated (C: open circles) or when heated just before radiation (C+heat: closed circles) and effects of thermotolerance (TT, squares). Note that TT (pre-heated long before radiation and thus without residual protein aggregates, but with high HSP levels show equal radiosensitivity (TT: open squares) as control cells, but reduced sensitization by the (2nd) test heat treatment (TT+heat: closed squares). In this schematic figure, data are corrected for cell death as induced by heat alone. Also, it is indicated how TERs are calculated. (d) Effect of hyperthermia on the sensitivity of cells to cisplatin (cDDP): comparison between a platin-responsive parental cell line (open symbols) and a cDDP-resistant variant derived from this cell line (closed symbols). After heating the cell lines have become equally cDDP responsive. Note that all figures (except (d)) are purely meant to be illustrative for educational purposes and that the axes are in arbitrary units and that the data do not represent actual data points. Data from (d) are redrawn from Hettinga et al. [21].

Temperature elevations transiently up-regulate a series of genes called heat shock genes that encode a class of proteins known as heat shock proteins (HSP). The mechanism responsible for the heat shock response is an autoregulatory loop: HSP normally keep the responsible transcription factor (HSF-1) inactive but upon heating HSP bind with higher affinity to unfolded proteins, triggering the release of HSF-1 from HSP which initiates HSP gene transcription. Once the protein damage/aggregation is restored after the heat shock by the HSP, substrate-free HSP themselves seem involved in attenuating the response by rebinding HSF-1 [10]. As a result, HSP levels transiently rise after heating but also gradually decline again upon prolonged stress free periods (Figure 1(a)). The up-regulation of HSP is closely associated with a transient resistant state of cells towards a subsequent second heat shock (thermotolerance: TT, Figure 1(a)). It is thought that the elevated HSP levels, by their chaperone activity (see below), protect cells against protein damage induced by the 2nd heating.

The HSP belong to the super family of proteins called molecular chaperones. Molecular chaperones are defined as proteins that bind to non-native or (partially) unfolded proteins and assist in their correct assembly by preventing their non-productive aggregation [11]. Under non-stressful circumstances, chaperones are involved in physiological processes in which proteins are on their way to their native structure (translation) or during which they need to be in a partially unfolded state (during transport over membranes). Under conditions of (thermal) stress, HSP can bind to denatured proteins and prevent their irreversible aggregation. By this binding, that still may lead to ‘controlled’ aggregation, the substrates are maintained in a state competent to be reactivated to an active protein or to be degraded upon stress relief. So protein aggregation can be reversible (Figure 1(a)) when HSP levels in cells are sufficiently high to cooperate with the damage. Elevating HSP prior to a heat shock, particular of the main chaperone HSP70 (e.g. by gene mediated transfection) indeed can induce heat resistance of cells [12–14] and this is linked to its ability to act as a chaperone [13], [14]. This, thus, confirms the relation between HSP expression in thermotolerant cells and the ability to deal with heat-induced protein damage [8].

Heat and radiation

Aggregation of nuclear proteins damage is thought to be the central event by which heat makes cells more sensitive to radiation [3], [15]. The synergy between heat and radiation, often expressed as Thermal Enhancement Ratios (TER: Figure 1(a, c)) is highest when the two modalities are given simultaneously. When heat precedes radiation, the synergy is lost when the time interval between the two modalities increases (Figure 1(b)) and this loss of TER nicely parallels the decline in protein aggregation [8] (Figure 1(a)). It is important to note that at the time when TER is maximal (during or immediately after heating) HSP levels have not yet increased; inversely, when HSP levels are maximal, cells have regained normal radiosensitivity (TER = 1; Figure 1(a, c)). This means that HSP are not involved in thermal radiosensitization and, more importantly, that physiological upregulations in HSP that make cells thermotolerant do not make them radioresistant (Figure 1(a, b)). However, when the thermotolerant cells are heated, they do become less well radiosensitized than non-TT control cells (Figure 1(b, c)) and the decline of radiosensitization is more rapid (Figure 1(b)) as if cells have been heated with a milder heat treatment. This is because in the TT cells nuclear protein aggregation is attenuated and/or repaired more rapidly due to the elevated HSP levels [8]. In other words, the HSP modulate the heat damage that is responsible for radiosensitization by heat, but they do not affect sensitivity to the radiation damage.

Whereas the decline in heat radiosensitization for heat given before radiation is, therefore, heat dose (or heat damage) dependent and prone to modulation by thermotolerance, the loss of interaction for radiation before heat is heat damage independent and solely dependent on the kinetics of repair DNA damage by the cells [15] (Figure 1(b)). If all DNA lesions are repaired before heating, no sensitization occurs. In fact these kinetics, together with the pioneer studies of Dewey and Sapareto [16] on chromosomal aberrations and many subsequent studies [15], [17] have led to the general consensus that thermal radiosensitization is due inhibition of repair of radiation-induced DNA damage. It is the current view that heat-induced repair inhibition is due to nuclear protein aggregation related effects on the (higher order) chromatin organization [15]. Genetic approaches have revealed that the functional presence of the two DNA DSB repair pathways, non-homologous end-joining and homologous recombination is not a pre-requisite for heat radiosensitization [17]. Based on lack of evidence for alternative mechanisms and based on some indirect biochemical evidence, it was therefore speculated [17] that heat effects on radiosensitivity may be mainly due to inhibition of the repolymerization step in base excision repair (BER). Although it is still unclear to what extent heat radiosensitization contributes to the interaction of heat and radiation in vivo, it is clear that thermo radiotherapy has been successful in several clinical trials (see [18] for overview).

Heat and drug interactions

A lot of physiology-related features make a combination of heat and drugs very attractive. Moreover, heat can cause more than additive killing when combined with alkylating agents, nitrosureas, platinum drugs and some antibiotics [19], although for some drugs only additive effects or even less than additive effects on cell death are found (Table I). It goes beyond this short overview to deal with all these drugs and heat interactions separately. Most impressive are data for heat and cisplatin treatments. Synergistic killing is already found at rather mild heat treatments [20]. Sensitization is multi-factorial and again heat-induced protein damage by heat may underlay these effects but the evidence for this is not yet clear. Heat enhances intra-cellular platinum accumulation, reduces intra-cellular detoxification leading to more platinum-induced DNA adducts and inhibits repair of these adducts [20]. Interestingly, all these features are up-regulated in cells that have acquired platinum resistance. This has led to the suggestion that hyperthermia could be instrumental in reversing drug resistance, a major clinical problem in chemotherapy. Indeed, several cisplatin-resistant cells can be made responsive to platinum again by combining the drug treatment with heat (Figure 1(d)), although only in certain cases resistance was found to be completely reversed [20]. Anyhow, the combination of chemotherapy with hyperthermia still requires further attention and has high potential, especially when combined with e.g. drug-targeting developments.

Table I.  Overview of the interaction between heat and some chemotherapeutic agents (derived from [19]).

Class of agents	Compounds tested	Interaction	Remarks
Alkylating agents	Melphalan; Ifosfamide; Mitomycin C	More than additive	Gradual increase with increasing temperature; Largest when simultaneous
Nitrosureas	BCNU; CCNU	More than additive	Largest when simultaneous
Platinum drugs	Cisplatin (cDDP); Carboplatin; Tetraplatin	More than additive	Gradual increase with increasing temperature; Largest when simultaneous
Antibiotics	Bleomycin	More than additive	Only >42°C; Largest when simultaneous
	Doxorubicin; Actinomycin D	Complex	Less than additive when heat precedes drug
Pyrimidine antagonists	5-FU; Ara-C	Additive
Vinca alkaloids	Vincristine; Vinblastine	Additive
Other	mAMSA; VP16	Complex	Less than additive when heat precedes drug