Kanwal Ahmed ( Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan. )
Syed Faisal Zaidi ( Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan. )
The fundamental idea and the effects of heat on cancer cells are well known. However, the results obtained in therapy by hyperthermia (HT) alone have been only partially satisfactory. Treatment at temperatures between 40 and 44°C is cytotoxic for cells in an environment with a low oxygen partial pressure and low pH, conditions that are found specifically within tumour tissue, due to insufficient blood perfusion. Under such conditions radiotherapy is less effective, and systemically applied cytotoxic agents will reach such areas in lower concentrations than in well-perfused areas. Therefore, clinically, it is preferred to use hyperthermia in combination with radiation therapy and chemotherapy. Hyperthermia can be applied by several methods: local hyperthermia by external or internal energy sources; regional hyperthermia by perfusion of organs or limbs, or by irrigation of body cavities; and whole-body hyperthermia. Number of studies have reported the combination of thermo-radiotherapy. Consequently, much attention has been focussed on identifying agents among the conventional chemotherapeutic substances that can sensitise tumour cells to hyperthermia-induced damage with minimal effects on normal cells. In this review, we overviewed important mechanisms of hyperthermia-induced apoptosis and the substances which can act as heat sensitisers in cancer therapy.
Keywords: Hyperthermia, Apoptosis, Heat sensitizer.
“Those who cannot be cured by medicine can be cured by surgery. Those who cannot be cured by surgery can be cured by fire [hyperthermia]. Those who cannot be cured by fire, they are indeed incurable”.—Hippocrates (479–377)
The use of elevated temperature, hyperthermia (HT) is not a new treatment for cancer. Hippocrates was aware of the potential of heat to cure or shrink tumours. Tumour shrinkage after a high fever due to an infection was reported in 1866.1 Heat has profound effects on cells. At low doses, heat enhances recovery from injury. At high doses, it leads to cell death that may be immediate for extreme doses. The role of HT alone as a cancer treatment is limited. It has been recognised as an effective and useful tool, especially in combination with conventional therapies to halt tumour growth. The significant advantage to this type of combination therapy is the possibility of using lower doses of chemotherapy and radiation leading to more effective treatment with fewer unwanted side effects, and a reduced resistance of cancer cells to the combination of drug and radiation. Numerous clinical trials have studied hyperthermia in combination with conventional therapies. Many of these studies, but not all, have shown a significant reduction in tumour size when hyperthermia is combined with other treatments.2 Temperature between 41°C-44°C is not toxic to the normal cells, but show toxicity in cancerous cells.
In the clinical application of HT, three methods can be employed: local, regional and whole-body HT. The choice of the method to use on any given patient depends on the type of the cancer, its location and its stage. Special thermometres are used to monitor the temperature during the course of the treatment.
Local HT is used to treat localised cancers. It can be applied by external, intraluminal and interstitial methods to treat tumours on the skin or just underneath it, tumours within or near the body cavities, and to treat deep tumours within the body, such as brain tumour. Different types of energy sources may be used to apply heat such as microwave, radiofrequency and ultrasound.3
Regional HT is used to heat large areas of tissue such as body cavity, organ or limb. Different methods can be employed depending on the type of cancer. For example, deep tissue regional HT is applied for cancers within the body like bladder4 or cervix; regional perfusion technique is applied for the cancers of arm and legs such as melanoma or cancers of some organs like liver or lung; peritoneal perfusion technique is applied for the treatment of cancers within the peritoneal cavity, including primary peritoneal mesothelioma and stomach cancer.3
Whole-body HT is used to treat metastatic cancers. A common characteristic is that energy is introduced into the body while at the same time energy losses are minimised. Most recent heat induction methods used are irradiation with infrared, radiofrequency and microwave electromagnetic energy. These techniques can be applied solely or in combination.3
Normal tissue toxicity will result directly from hyperthermia when the tolerance limits are exceeded. Experimental studies have shown that most normal tissues are not damaged when the temperature over 1 hour of treatment does not exceed 44°C. The toxicity from superficial hyperthermia is usually a skin burn, heals with conservative treatment. Subcutaneous fat burns were seen in 3%-12% of the patients treated with deep hyperthermia. It does not usually cause much discomfort to the patient who feels a subcutaneous lump, which is tender for a few days to a maximum of a few weeks and then disappears spontaneously. Toxicity from whole-body hyperthermia depends on, besides temperature, the patient’s general condition, condition of organ systems and the physiological conditions during the treatment. During any application of hyperthermia, it is important to avoid pressure sites, since hypoxic normal tissues will be more sensitive to hyperthermia.3
HT is known as a potent radio-sensitiser and there are many studies which deal with the biochemical and biophysical mechanisms leading to cell death from HT or from a combination of HT and ionising radiation. Studies are also available on the combination of HT and drugs-induced apoptosis. In this review, we will discuss the factors and mechanisms which make combination of HT with drugs and radiation an effective tool for cancer therapy.
Cellular physiological changes induced by HT
Hyperthermia induces numerous changes in cellular physiology (Table-1).
These cellular alterations make a combination of heat with drugs and radiation very effective. Below some important cellular responses to potential therapeutic regimens are discussed in detail:
Alteration in membrane permeability
Membranes are known to be extremely sensitive to heat stress because of their complex molecular composition of lipids and proteins. At a certain temperature, lipids change from a tightly packed gel phase to a less tightly packed crystalline phase, and permeability of the cell membrane (membrane fluidity) increases. Hyperthermia-induced cell membrane permeability leads to increased drug delivery into tumour cells. In addition, increased vascular permeability due to heat increases in endothelial gap size also aids drug delivery into the tumours. Alteration in membrane permeability also alters the cellular content of several ions (Na+, Mg2+, K+) in a number of cells, although the changes in these ion balances are not primarily responsible for hyperthermic cell death.5 Another ion which might be involved in hyperthermic cell death is Ca2+. Influxes of extracellular Ca2+ stimulate the activity of calmodulin-dependent protein kinases, inositol triphosphate production and other signalling cascades.6
Modification of cytoskeletal system
The response of cytoskeletal systems to HT varies depending on the cell type and HT dose.7 Hyperthermia-induced disassembly of the cytoskeleton, enlarges the tumour pores which enables easier drug delivery.7 It also induces alteration of the mitotic spindles,7 centrosome organisation, and protein denaturation which results in the formation of multinucleated non-clonogenic cells.8 Furthermore, it also induces alterations of integrin-cytoskeleton network with concomitant cell shape change, anoikis and programmed cell death.9
Inhibition of DNA repair
Dioxyribonucleic acid (DNA) appears to be the primary target for most of the currently adopted chemotherapeutic drugs and radiation.10 The main obstacle for cancer treatment is the resistance of cells to the cytotoxic effects of drugs and radiation. Several possible mechanisms have been suggested to account for this resistance, for example, intrinsic DNA repair capabilities, vascular insufficiency, and cellular impermeability.11 Hyperthermia is reported to induce DNA double-strand breaks due to the denaturation and dysfunction of heat-labile repair proteins such as DNA polymerases12 or to the precipitation of denatured proteins onto nuclear chromatin structures, generating a barrier which prevents repair enzymes from reaching damage sites.13 HT-induced protein denaturation is also reported to alter multiple nuclear matrix-dependent functions [e.g., DNA replication, DNA transcription, Messengar ribonucleic acid (mRNA) processing and DNA repair].14
It is thus hypothesised that hyperthermia treatment sensitises tumour cells to chemotherapeutic drugs or radiation by altering cytoskeleton re-organisation, enhancing membrane permeability and inhibiting DNA repair.15
HT-induced signalling pathways
Pro-apoptotic signaling pathways
Apoptosis is a genetically programmed and biochemically active mode of cell death in which the cell actively participates in its own destruction.16 It is required for cell life span regulation and normal development.15 Apoptosis also aids in the self-deletion of injured cells, terminal differentiation of epithelial cells, and organ and tissue shaping.16 Abnormalities in this process are implicated in several human diseases, including cancer. Radiation, cytotoxic drugs, viruses and hyperthermia can trigger this process.
Hyperthermia within a temperature range of 41-45ºC induces apoptosis to varying degrees in many cell lines.17 It induces apoptosis mainly through reactive oxygen species (ROS) generation, and a likely source of elevated ROS production is the mitochondria.17 Mitochondria are believed to produce basal levels of ROS in the form of single-electron leakage to oxygen during normal metabolism.18 This can increase dramatically under conditions in which the mitochondria are damaged or exposed to certain toxic conditions.17 In addition, HT can also alter the expression of the Bax and Bcl-2 genes, where such changes are dependent on the sensitivity of cell lines to HT.19 In thermo-resistant cell lines, HT by itself cannot change the expression of Bax and Bcl-2, but the combination of HT and chemotherapy or radiotherapy can up-regulate Bax and down-regulate Bcl-2 expression.20 Furthermore, hyperthermia-induced increase in intracellular Ca2+ ion ([Ca2+]i) concentration is also thought to be involved in cell death. However, evidence for the role of [Ca2+]i in hyperthermic cell death is contradictory. Some investigators have stated that HT-induced increase in Ca2+ does not play a key role,21 while others have concluded that thermal perturbations in [Ca2+]i are among the primary events leading to heat-induced cell killing.22 These observations suggest that the role of Ca2+ in HT-induced cell killing may be dependent on cell type. HT is reported to increase the expression of the receptor inositol triphosphate23 which may regulate the release of Ca2+. Generally, Ca2+ can act on multiple targets to trigger apoptosis.24 Lipid peroxidation due to ROS generation is also reported to alter Ca2+ distribution25 and to activate a Ca2+-dependent apoptotic pathway23 (Figure).
Anti-apoptotic signaling pathways
HT is also responsible to activate survival/antiapoptotic pathways that lead to the development of thermo tolerance. Signalling factors, such as Akt, p38, extracellular signal-regulated kinase (ERK) etc play important roles in anti-apoptosis or cellular proliferation pathways.26 Such anti-apoptosis and cytoprotective signalling factors are negative for hyperthermic cancer therapy. Therefore, targeted inhibition of the anti-apoptotic signalling pathways is an attractive approach for the development of hyperthermia-induced chemo-sensitisation.
Heat shock proteins (HSPs)
Heat shock proteins (HSPs) were originally identified as proteins whose expression was markedly increased by heat shock.26 HSPs represent a heterogeneous group of molecular chaperones consisting of at least five sub-groups with different molecular mass and partially varying biological function. HSPs are usually divided into small HSPs (molecular mass <40 kDa), and the HSPs60, HSPs70, HSPs90 and HSPs100 proteins families.26 All HSPs families share their chaperoning function, i.e. they unselectively bind to hydrophobic protein sequences liberated by denaturation. Thus they prevent irreversible interaction with neighbour proteins that would result in a loss of function. Several HSPs are expressed even in unstressed cells and play important function in normal cell physiology.26 HSPs synthesis can be induced within minutes by activation of so-called ‘heat shock factors’(HSF). In hyperthermia, HSPs are thought to be involved in the protection of cells against heat damage.
Hyperthermia and drug resistance
Drug resistance represents the major cause of treatment failure in human malignancies, and can be induced by different mechanisms, of which the pleitrope multidrug resistance (MDR), has gained particular interest.27 Hyperthermia is a good candidate to overcome various modes of drug resistance. A number of phase I and II trials reported successful treatments of patients with chemorefractory tumours by adding HT to antineoplastic chemotherapy.28 On the other hand, moderate heat exposure has been demonstrated to induce HSP expression in cultured cells, and elevated levels of intracellular HSP 70 have been shown to be associated with thermotolerance which may be linked with different forms of drug resistance (e.g. MDR), and HT has been shown to induce various forms of drug resistance, too (including MDR or the heat-dependant inactivation of the enzyme topoisomerase II). Reversal of drug resistance has been particularly shown for platinum compounds at temperatures >42°C, whereas induction of drug resistance (alone or in conjunction with thermotolerance and HSP-accumulation) may appear when lower temperatures are applied. However, available data on hyperthermia and drug resistance suggest that the positive effect (reversal of drug resistance) overweighs the disadvantages (induction of drug resistance) in clinical practice.29
Hyperthermia and radiation act in a synergistic way. This synergism induces an increase in cell killing even at lower temperatures, which is not the case when hyperthermia is implemented alone.29 It appears most pounced in S-phase cells that are usually resistant to radiation alone. Hypoxic cells, as well as, those with impaired nutrient supply and/or acidic pH have been shown to react very sensitively to the combined treatment of heat and radiation.30
Synthetic heat sensitisers:
One of the promising approaches to the clinical application of HT is to combine it with chemotherapy (‘thermochemotherapy’). Numerous in vitro and in vivo systems have been used to demonstrate the synergistic effects of HT when combined with a wide range of chemotherapeutic agents. The combined treatments have shown increased success in many tumours.31 The synergistic effects occur due to the alteration in the pharmacokinetics32 and pharmacodynamics of drugs,29 increases DNA damage11 and decreases DNA repair.5,8 It converts some innocuous drugs into highly toxic agents the so-called ‘heat sensitisers/thermosensitisers.’ An ideal heat sensitiser would be non-toxic at normal temperatures, but become toxic at hyperthermic temperature. There are chemical agents that can act as heat sensitisers at non-toxic concentrations (Table-2).
Natural heat sensitisers
Natural compounds contain greater characteristics of high chemical diversity and biochemical specificity than standard chemical compounds. Several studies have reported the role of natural compounds in cancer cell apoptosis.33 However, quercetin is the only bioflavonoid reported in combination with hyperthermia as an inhibitor of HSP 70.34Numbers of medicinal plants are present which can be studied with HT in order to identify effective heat sensitisers for cancer therapy.
The clinical application of HT has a strong biological rationale. Still research is going on to overcome the technical problems associated with this therapy. Resistance to conventional anti-cancer drugs creates a dire need to apply alternative strategies like HT in treating cancers. Furthermore, combination of HT with available chemotherapeutic agents or with novel candidates from natural source might open new dimensions in targeting resistant cancers. Further elucidation of the molecular mechanisms responsible for the enhancement of heat-induced apoptotic cell death may provide a strong basis for the effective use of HT and HT-induced apoptosis sensitisers in cancer therapy.
1. DeNardo GL, DeNardo SJ. Update: Turning the heat on cancer. Cancer Biother and Radiopharm 2008; 23: 671-80.
2. Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T, et al. The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol 2002; 43: 33-56.
3. van der Zee J. Heating the patient: a promising approach? Ann Oncol 2002; 13: 1173-84.
4. Rampersaud EN, Vujaskovic Z, Inman BA. Hyperthermia as a treatment for bladder cancer. Oncology (Williston Park) 2010; 24: 1149-55.
5. Ruifork AC, Kanon B, Konings AW. Heat induced K2+ loss, trypan blue uptake and cell lysis in different cell lines: effect of serum. Radiat Res 1987; 109: 303-9.
6. Cividalli A, Cruciani G, Livdi E, Pasqualetti P, Tirindelli Danesi D. Hyperthermia enhances the response of paclitaxel and radiation in a mouse adenocarcinoma. Int J Radiat Oncol Biol Phys 1999; 44: 407-12.
7. Huang SH, Yang KJ, Wu JC, Chang KJ, Wang SM. Effects of hyperthermia on the cytoskeleton and focal adhesion proteins in a human thyroid carcinoma cell line. J Cell Biochem 1999; 75: 327-37.
8. Vidair CA, Doxsey SJ, Dewey WC. Heat shock alters centrosome organizationleading to mitotic dysfunction and cell death. J Cell Physiol 1993; 154: 443-55.
9. Yonezawa M, Otsuka T, Matsui N, Tsuji H, Kato KH, Moriyama A, et al. Hyperthermia induces apoptosis in malignant fibrous histiocytoma cells in vitro. Int J Cancer 1996; 66: 347-51.
10. Kohn KW. DNA as a target in cancer chemotherapy: Measurement of macromolecular DNA damage produced in mammalian cells by anticancer agents and carcinogens. Methods in Cancer Research. New York, Academic 1979; 16: 291-345.
11. DeVita JrVT, Principles of chemotherapy. (Eds. VT DeVita, S Hellman, SA Rosenberg) In: ‘Cancer Principles and Practice of Oncology. 2nd ed. Philadelphia: Lippincott 1985; pp 257-86.
12. Takahashi A, Matsumoto H, Kosuke N, Kitano M, Hirose S, Tanaka H, et al. Evidence for the involvement of double-strand breaks in heat-induced cell killing. Cancer Res 2004; 64: 8839-45.
13. Wachsberger PR, Iliakis G. Hyperthermia does not affect rejoining of DNA double-strand breaks in a cell free assay. Int J Radiat Biol 2000; 76: 313-26.
14. Tentori L, Orlando L, Lacal PM, Benincasa E, Faraoni I, Bonmassar E, et al. Inhibition of O6-alkylguanine DNA-alkyltransferase or poly (ADP-ribose) polymerase increases susceptibility of leukemic cells to apoptosis induced by temozolomide. Mol Pharmacol 1997; 52: 249-58.
15. Luchetti F, Mannello F, Canonico B, Battistelli M, Burattini S, Falcieri E, et al. Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis. Apoptosis 2004; 9: 635-48.
16. Poe B, O’Neill K. Inhibition of protein synthesis sensitizes thermotolerant cells to heat shock-induced apoptosis. Apoptosis 1997; 2: 510-7.
17. Zuo L, Christofi FL, Wright VP, Liu CY, Merola AJ, Berliner LJ. Intra and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle. Am J Physiol Cell Physiol 2000; 279: C1058-C66.
18. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59: 527-605.
19. Basile A, Biziato D, Sherbet GV, Comi P, Cajone F. Hyperthermia inhibits cell proliferation and induces apoptosis: relative signaling status of P53, S100A4, and notch in heat sensitive and resistant cell lines. J Cell Biochem 2008; 103: 212-20.
20. Liang H, Zhan JH, Wang BG, Pan Y, Hao XS. Change in expression of apoptosis genes after hyperthermia, chemotherapy and radiotherapy in human colon cancer transplanted into nude mice. World J Gastroentrol 2007; 13: 4365-71.
21. Vidair CA, Wang ZH, Dewey WC. Non-involvement of heat-induced increase in intracellular free Ca2+ concentration for thermal killing and induction of thermotolerance. Radiat Res 1990; 124: 156-64.
22. Mikkelsen RB, Reinlib L, Donowitz M, Zahniser D. Hyperthermia effects on cytosolic [Ca2+]. Analysis at single cell level by digitized imaging microscopy and cell survival. Cancer Res 1991; 51: 359-64.
23. Li FJ, Kondo T, Zhao QL, Tanabe K, Ogawa R, Li M, et al. Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2’-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells. Free Radiat Res 2001; 35: 281-99.
24. Nunez G, Benedict MA, Hu Y, Inohara N. Caspases: the proteases of the apoptotic pathway. Oncogene 1998; 17: 3237-45.
25. Fabisiak JP, Tyurina YY, Tyurin VA, Lazo JS, Kagan VE. Random versus selective membrane phospholipid oxidation in apoptosis: Role of phosphatidylserine, Biochemistry 1998; 37: 13781-90.
26. Ohnishi K, Ohnishi T. Hyperthermic sensitizers targeting heat-induced signal transduction. Ann Cancer Res Therap 2007; 15: 35-40.
27. Hildebrandt B, Wust P. Interaction between hyperthermia and cytotoxic drugs. Cancer Treat Res 2007; 134: 185-93.
28. Hildebrandt B, Hegewisch-Becker S, Kerner T, Nierhaus A, Bakhshandeh-Bath A, Janni W, et al Current status of radiant whole-body hyperthermia at temperatures > 41.5°C and practical guidelines for the treatment of adults. The German “Interdisciplinary Working Group on Hyperthermia”. Int J Hyperthermia 2005; 21: 169-83.
29. Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T et al. The cellular and molecular basis of hyperthermia. Crit Rev Oncol/Hematol 2002; 43: 33-56.
30. Dewey WC. Arrhenius relationships from the molecule and cell to the clinic. Int J Hyperthermia 1994; 10: 457-83.
31. Li GC. Thermal biology and physiology in clinical hyperthermia: current status and Future needs. Cancer Res 1984; 44: 4886s-93s.
32. Bull JM. An update on the anticancer effects of a combination of chemotherapy and hyperthermia. Cancer Res 1984; 44: 4853s-6s.
33. Zhang Z, Knobloch TJ, Seamon LG, Stoner GD, Cohn DE, Paskett ED, et al. A black raspberry extract inhibits proliferation and regulates apoptosis in cervical cancer cells. Gynecol Oncol 2011; 12: 401-6.
34. Ramasamy S, Abdul Wahab N, Zainal Abidin N, Manickam S. Effect of extracts from Phyllanthus watsonii Airy Shaw on cell apoptosis in cultured human breast cancer MCF-7 cells. Exp Toxicol Pathol 2012 (Epub ahead of print).
35. Ahmed K, Hori T, Yu DY, Wei ZL, Zhao QL, Nakashima M, et al. Hyperthermia chemo-sensitization, chemical thermo-sensitization and apoptosis. Thermal Med 2008; 24: 1-12.