Hyperthermia-induced Cell Death Research Articles

Induction heating: an efficient methodology for the synthesis of functional core-shell nanoparticles.

Induction heating has been applied for a variety of purposes over the years, including hyperthermia-induced cell death, industrial manufacturing, and heterogeneous catalysis. However, its potential in materials synthesis has not been extensively studied. Herein, we have demonstrated magnetic induction heating-assisted synthesis of core-shell nanoparticles starting from a magnetic core. The induction heating approach allows an easy synthesis of FeNi3@Mo and Fe2.2C@Mo nanoparticles containing a significantly higher amount of molybdenum on the surface than similar materials synthesized using conventional heating. Exhaustive electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy characterization data are presented to establish the core-shell structures. Furthermore, the molybdenum shell was transformed into the Mo2C phase, and the catalytic activity of the resulting nanoparticles tested for the propane dry reforming reaction under induction heating. Lastly, the beneficial role of induction heating-mediated synthesis was extended toward the preparation of the FeNi3@WOx core-shell nanoparticles.

Modelling the effect of vascular status on tumour evolution and outcome after thermal therapy

Microscale oxygenation plays a prominent role in tumour progression. Spatiotemporal variability of oxygen distribution in the tumour microenvironment contributes to cellular heterogeneity and to the emergence of normoxic and hypoxic populations. Local levels of oxygen strongly affect the response of tumours to the administration of different therapeutic modalities and, more generally, to the phenomenon of resistance to treatments. Several interventions have been proposed to improve tumour oxygenation, being the elevation of the local temperature (hyperthermia) an important one. While other factors such as the metabolic activity have to be considered, the proficiency of the tumour vascular system is a key factor both for the tissue oxygenation and for its temperature maps. Consequently, the interplay of these factors has attracted considerable attention from the mathematical modelling perspective. Here we put forward a transport-based system of partial differential equations aimed at describing the dynamics of healthy and tumour cell subpopulations at the microscale in a region placed between two blood vessels. By using this model with diverse flow conditions, we analyse the oxygen and temperature profiles that arise in different scenarios of vascular status, both during free progression and under thermal therapy. We find that low oxygen levels are associated to elevations of temperature in locations preferentially populated by hypoxic cells, and hyperthermia-induced cell death, being strongly dependent on blood flow, would only appear under highly disrupted conditions of the local vasculature. This results in a noticeable effect of heat on hypoxic cells. Additionally, when pronounced cell death occurs, it is followed by a significant increase in the oxygen levels. Our results provide quantitative insight to the physiological and biological processes taking place at sub-voxel sizes, currently not accessible to standard functional imaging due to spatial resolution limitations.

Hyperthermia-induced Cell Death

Hyperthermia-induced Cell Death

Ethanol Enhances Hyperthermia-Induced Cell Death in Human Leukemia Cells.

Ethanol has been shown to exhibit therapeutic properties as an ablative agent alone and in combination with thermal ablation. Ethanol may also increase sensitivity of cancer cells to certain physical and chemical antitumoral agents. The aim of our study was to assess the potential influence of nontoxic concentrations of ethanol on hyperthermia therapy, an antitumoral modality that is continuously growing and that can be combined with classical chemotherapy and radiotherapy to improve their efficiency. Human leukemia cells were included as a model in the study. The results indicated that ethanol augments the cytotoxicity of hyperthermia against U937 and HL60 cells. The therapeutic benefit of the hyperthermia/ethanol combination was associated with an increase in the percentage of apoptotic cells and activation of caspases-3, -8 and -9. Apoptosis triggered either by hyperthermia or hyperthermia/ethanol was almost completely abolished by a caspase-8 specific inhibitor, indicating that this caspase plays a main role in both conditions. The role of caspase-9 in hyperthermia treated cells acquired significance whether ethanol was present during hyperthermia since the alcohol enhanced Bid cleavage, translocation of Bax from cytosol to mitochondria, release of mitochondrial apoptogenic factors, and decreased of the levels of the anti-apoptotic factor myeloid cell leukemia-1 (Mcl-1). The enhancement effect of ethanol on hyperthermia-activated cell death was associated with a reduction in the expression of HSP70, a protein known to interfere in the activation of apoptosis at different stages. Collectively, our findings suggest that ethanol could be useful as an adjuvant in hyperthermia therapy for cancer.

3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments

For multimodality therapies such as the combination of hyperthermia and radiation, quantification of biological effects is key for dose prescription and response prediction. Tumour spheroids have a microenvironment that more closely resembles that of tumours in vivo and may thus be a superior in vitro cancer model than monolayer cultures. Here, the response of tumour spheroids formed from two established human cancer cell lines (HCT116 and CAL27) to single and combination treatments of radiation (0–20 Gy), and hyperthermia at 47 °C (0–780 CEM43) has been evaluated. Response was analysed in terms of spheroid growth, cell viability and the distribution of live/dead cells. Time-lapse imaging was used to evaluate mechanisms of cell death and cell detachment. It was found that sensitivity to heat in spheroids was significantly less than that seen in monolayer cultures. Spheroids showed different patterns of shrinkage and regrowth when exposed to heat or radiation: heated spheroids shed dead cells within four days of heating and displayed faster growth post-exposure than samples that received radiation or no treatment. Irradiated spheroids maintained a dense structure and exhibited a longer growth delay than spheroids receiving hyperthermia or combination treatment at (thermal) doses that yielded equivalent levels of clonogenic cell survival. We suggest that, unlike radiation, which kills dividing cells, hyperthermia-induced cell death affects cells independent of their proliferation status. This induces microenvironmental changes that promote spheroid growth. In conclusion, 3D tumour spheroid growth studies reveal differences in response to heat and/or radiation that were not apparent in 2D clonogenic assays but that may significantly influence treatment efficacy.

Tumor-specific delivery of gemcitabine with activatable liposomes

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine‑copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

Mechanistic study of nonivamide enhancement of hyperthermia-induced apoptosis in U937 cells.

Hyperthermia is one therapeutic tool for damaging and killing cancer cells, with minimal injury to normal tissues. However, its cytotoxic effects alone are insufficient for quantitative cancer cell death. To overcome this limitation, several studies have explored non-toxic enhancers for hyperthermia-induced cell death. Capsaicin may be applicable as a therapeutic tool against various types of cancer. In the present study, we employed nonivamide, a less-pungent capsaicin analogue, to investigate its possible enhancing effects on hyperthermia-induced apoptosis; moreover, we analyzed its molecular mechanism. Treatment of U937 cells at 44 °C for 15 min, combined with nonivamide 50 μM, revealed enhancement of apoptosis. Significant increases in reactive oxygen species generation, mitochondrial dysfunction, and cleaved caspase-3 were observed during the combined treatment; these were accompanied by an increase in pro-apoptotic Bcl-2 family proteins and a decrease in anti-apoptotic Bcl-2 proteins. In addition, significant increases in p-JNK and p-p38 were detected, following the combined treatment. In conclusion, nonivamide enhanced hyperthermia-induced apoptosis via a mitochondrial-caspase dependent pathway. The underlying mechanism may include elevation of intracellular reactive oxygen species, mitochondrial dysfunction, and increased activation of JNK and p38.

HSPB1 deficiency sensitizes melanoma cells to hyperthermia induced cell death.

Hyperthermia has shown clinical potency as a single agent or as adjuvant to other therapies in cancer treatment. However, thermotolerance induced by thermosensitive genes such as the heat shock proteins can limit the efficacy of hyperthermic treatment. In the present study, we identified HSPB1 (HSP27) is hyperthermically inducible or endogenously highly expressed in both murine and human melanoma cell lines. We used a siRNA strategy to reduce HSPB1 levels and showed increased intolerance to hyperthermia via reduced cell viability and/or proliferation of cells. In the investigation of underlying mechanisms, we found knock down of HSPB1 further increased the proportion of apoptotic cells in hyperthermic treated melanoma cells when compared with either single agent alone, and both agents leaded to cell cycle arrest at G0/G1 or G2/M phases. We concluded that hyperthermia combined with silencing of HSPB1 enhanced cell death and resulted in failure to thrive in melanoma cell lines, implying the potential clinical utility of hyperthermia in combination with HSPB1 inhibition in cancer treatment.

Abstract 2756: SMARCAD1 depletion enhances hyperthermia-mediated radiosensitization by decreasing resection and enhancing stalled replication forks

Abstract Hyperthermia is the most effective chemo- and radio-sensitizer in use clinically for containing tumor growth. Hyperthermia is proposed to induce degradation of BRCA2, the protein involved in homologous recombination (HR) repair of DNA double strand breaks and, thus sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition. We and other laboratories have reported that hyperthermia inhibits DNA damage repair. Since HR includes single-strand annealing, gene conversion and break-induced replication, it is critical for DNA replication during recovery of stalled replication forks, but it is not yet clear how hyperthermia affects these important steps in HR. Here we report that SMARCAD1 depletion increases hyperthermia induced cell death and chromosome damage in irradiated S-phase cells. Combined SMARCAD1 depletion and hyperthermia treatment decreased repair and recruitment of repairosome proteins to ISce-1 induced DSBs as well as decreased IR-induced repairosome foci formation. This effect was limited to S-phase cells, where homologous recombination is up regulated and the predominant pathway of DSB repair. Hyperthermia decreased the quantity of 5′ single stranded DNA intermediates at endonuclease-generated DNA break sites, an effect that was further decreased by SMARCAD1 depletion. Consistent with these results, simultaneous SMARCAD1 depletion and hyperthermia increased replication fork stalling and affected firing of new replication origins. The results presented here support a mechanism by which SMARCAD1 depletion and hyperthermia mediate radiosensitization through inhibition of the DNA resection step that precedes strand homology search during HR repair of IR-induced DSBs. Citation Format: Sharmistha Chakraborty, Clayton R. Hunt, Shashank Hambarde, Kalpana Mujoo, Nobuo Horikoshi, Raj K. Pandita, Abid Mattoo, Bin Teh, Tej K. Pandita. SMARCAD1 depletion enhances hyperthermia-mediated radiosensitization by decreasing resection and enhancing stalled replication forks. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2756.

A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles

Plasmonic nanoparticles are increasingly utilized in biomedical applications including imaging, diagnostics, drug delivery, and plasmonic photothermal therapy (PPT). PPT involves the rapid conversion of light into heat by plasmonic nanoparticles targeted to a tumor, causing hyperthermia-induced cell death. These nanoparticles can be passively targeted utilizing the enhanced permeability and retention effect, or actively targeted using proteins, peptides, or other small molecules. Here, we report the use of actively targeted spherical gold nanoparticles (AuNPs), both to induce PPT cell death, and to monitor the associated molecular changes through time-dependent surface enhanced Raman spectroscopy within a single cell. We monitored these changes in real-time and found that heat generated from the aggregated nanoparticles absorbing near-infrared (NIR) laser light of sufficient powers caused modifications in the protein and lipid structures within the cell and ultimately led to cell death. The same molecular changes were observed using different nanoparticle sizes and laser intensities, indicating the consistency of the molecular changes throughout PPT-induced cell death from actively targeted AuNPs. We also confirmed these observations by comparing them to reference spectra obtained by cell death induced by oven heating at 100 °C. The ability to monitor PPT-induced cell death in real-time will help understand the changes on a molecular level and offers us a basis to understand the molecular mechanisms involved in photothermal cancer cell death.

Moderate Hyperthermia Induces Apoptosis in Metaphase-Arrested Cells But Not in Interphase Hela Cells

Metaphase-arrest agents and hyperthermia are both known to be capable of inducing apoptosis, and they have been used, separately, in cancer treatments. Here, we have examined whether the two treatments together may have a synergistic effect. We find that when H-HeLa cells are arrested in metaphase with spindle poisons (nocodazole or paclitaxel) and then subjected to mild heat treatment (41.5℃), they exhibit morphological changes typical of apoptosis within three hours. Moreover, those changes are blocked by the pan-caspase inhibitor zVAD-fmk, indicating apoptosis, and activated Procaspase 3 is detected by immunoblotting and by staining with the fluoresce-in-labelled caspase inhibitor FAM-VAD-fmk. Interphase cells treated in the same way do not under-go apoptosis, even with spindle poisons present. Induction of apoptosis is more rapid when the cells have been arrested longer in metaphase, suggesting that accumulation or depletion of some cellular component(s) during metaphase-arrest may make them more susceptible to hyperthermia. Further work is in progress to test whether other cell lines exhibit the same behavior and to learn more about the mechanism. The phenomenon is of interest because it may provide clues to how hyperthermia induces cell death and may yield novel therapeutic approaches to block or stimulate apoptosis.

Thermotolerance induced at a mild temperature of 40 °C alleviates heat shock-induced ER stress and apoptosis in HeLa cells

Hyperthermia (39–45°C) has emerged as an alternate prospect for cancer therapy in combination with radiation and chemotherapy. Despite promising progress in the clinic, molecular mechanisms involved in hyperthermia-induced cell death are not clear. Hyperthermia causes protein denaturation/aggregation, which results in cell death by apoptosis and/or necrosis. Hyperthermia also induces thermotolerance, which renders cells resistant to subsequent exposure to lethal heat shock. This study investigates the role of both lethal (42–43°C) and mild (40°C) hyperthermia in regulating ER stress and ER stress-induced apoptosis in HeLa cells. The ability of mild thermotolerance induced at 40°C to alleviate either or both of these processes is also determined. Hyperthermia (42–43°C) induced ER stress, revealed by phosphorylation of PERK, eIF2α and IRE1α, cleavage of ATF6 and increased expression of BiP and sXBP1. Real-time PCR revealed that mRNA levels of ATF6, ATF4, BiP, sXBP1 and CHOP increased in cells exposed to hyperthermia. Moreover, hyperthermia caused disruption of calcium homeostasis and activated the calpain-calpastatin proteolytic system and ER resident caspase 4. Pre-exposure to mild hyperthermia (40°C) alleviated the induction of cytotoxicity and ER stress by hyperthermia (42–43°C) and protected cells against ER stress-induced apoptosis. ShRNA-mediated depletion of Hsp72 abrogated protective effects of mild thermotolerance (40°C) against heat-shock induced ER stress and sensitized cells to ER stress-mediated apoptosis. Our findings show that Hsp72 contributes to the protective effects of mild hyperthermia (40°C) against hyperthermia-induced ER stress and apoptosis.

Molecular mechanisms of hyperthermia-induced apoptosis enhanced by docosahexaenoic acid: Implication for cancer therapy

To develop a non-toxic enhancer for hyperthermia-induced cell death as a potential cancer treatment, we studied the effect and mechanism of docosahexaenoic acid (DHA) on hyperthermia-induced apoptosis. Treatment with 20μM DHA and 44°C for 10min induced significant apoptosis, increased intracellular reactive oxygen species (ROS), and caspase-3 activation in U937 cells, but heat or DHA alone did not induce notable apoptosis. Decreased mitochondrial transmembrane potentials were dramatically increased by the combined treatment, accompanied by increased pro-apoptotic Bcl-2 family protein tBid, and decreased anti-apoptotic Bcl-2 and Bcl-xL. Combined hyperthermia–DHA treatment induced significant phosphorylation of protein kinase C (PKC)-δ (p-PKC-δ), and apoptosis in a DHA dose-dependent manner. Using both 20μM DHA and 44°C for 10min induced significant PKC-δ cleavage and its translocation to mitochondria. These results were also seen in HeLa cells. However, MAPKs and Akt were not affected by the treatment. In conclusion, DHA enhances hyperthermia-induced apoptosis significantly via a mitochondria–caspase-dependent pathway; its underlying mechanism involves elevated intracellular ROS, mitochondria dysfunction, and PKC-δ activation.

The Effect of Mild Hyperthermia on Morphology, Ultrastructure and F-Actin Organization in HL-60 Cell Line

Introduction. Hyperthermia is a well-established physical stimulus, which is applied as an adjunctive therapy with various cancer treatments, such as radiotherapy and chemotherapy. However, the precise mechanism of heat action at the cellular level remains to be elucidated, and appears to be multi-dimensional. The purpose of the current study was to determine the effect of mild hyperthermia on the actin cytoskeleton in the HL-60 cell line. In addition, the morphological and ultrastructural approaches were used to determine the type of hyperthermia-induced cell death. Material and methods. All studies were performed using human promyelocytic leukemia cell line (HL-60). Actin filaments were visualized with phalloidin conjugated to Alexa Fluor® 488 using fluorescence microscopy. Morphological and ultrastructural changes in the HL-60 cells were analysed by light and electron microscopy, respectively. Results. Exposure of HL-60 cells to mild hyperthermia resulted in the reorganization of the actin cytoskeleton and the appearance of characteristic apoptotic features, including cell shrinkage, chromatin condensation and margination. In addition, swollen mitochondria were observed. The morphological and ultrastructural changes increased in severity with an increase in recovery time. Similarly, actin filament remodeling was observed immediately after the heat shock and was more evident 3 and 6 hrs after the treatment. These effects were mainly reflected by a higher definition of the dense cortical F-actin ring as well as the appearance of brightly fluorescent F-actin dots and networks scattered throughout the cytoplasm. Conclusions. Presented data suggest that actin filament reorganization is involved in the process of apoptosis initiated by mild hyperthermia. Furthermore, the results of our studies showed that the severity of hyperthermia-induced morphological and ultrastructural changes as well as alterations in actin organization depend not only on the temperature treatment but also on the duration of post heat shock recovery

Temperature‐dependent vascular endothelial growth factor (VEGF) induction in human retinal pigment epithelium – implications for transpupillary thermotherapy in uveal melanoma

AbstractPurpose Transpupillary thermotherapy is a hyperthermia treatment for small uveal melanomas but its use is controversially discussed. In uveal melanomas, vascular endothelial growth factor (VEGF) increase is correlated with metastases, and VEGF increase can be found in treated melanomas. In this study, the effect of hyperthermia on the VEGF secretion in human RPE cells was studied.Methods Immortal human retinal pigment epithelium (RPE) cell line Arpe‐19 was exposed to 40°, 42°, 45° and 50°C for 1 min, 5 min and 15 min. Toxicity was evaluated using trypan blue exclusion assay and VEGF secretion was evaluated by ELISA. Involvement of Mitogen activated protein kinase (MAPK) pathways and Transient Receptor Potential Vanilloid (TRPV) channels on VEGF induction was investigated using commercially available inhibitors.Results Hyperthermia induces cell death in a time and temperature dependent manner. VEGF expression and secretion is strongly induced by hyperthermia in a time and temperature dependent manner. VEGF induction is mediated by p38 and to a lesser degree by JNK. TRPV channels are only involved in VEGF secretion at lower temperatures.Conclusion Hyperthermia induces a temperature dependent secretion of VEGF in human RPE, which is mediated by p38. As VEGF may be involved in the development of micrometastases, these findings indicate that thermotherapy for the treatment of uveal melanomas should be regarded with caution.

Stability of antibody-conjugated gold nanoparticles in the endolysosomal nanoenvironment: implications for noninvasive radiofrequency-based cancer therapy

The use of noninvasive radiofrequency (RF) electric fields as an energy source for thermal activation of nanoparticles within cancer cells could be a valuable addition to the emerging field of nano-mediated cancer therapies. Based on investigations of cell death through hyperthermia, and offering the ability for total-body penetration by RF fields, this technique is thought to complement and possibly outperform existing nano-heat treatments that utilize alternative heat production via optical or magnetic stimuli. However, it remains a challenge to understand fully the complex RF-nanoparticle-intracellular interactions before full system optimization can be engineered. Herein we have shown that liver cancer cells can selectively internalize antibody-conjugated gold nanoparticles (AuNPs) through receptor-mediated endocytosis, with the nanoparticles predominantly accumulating and aggregating within cytoplasmic endolysosomes. After exposure to an external RF field, nonaggregated AuNPs absorbed and dissipated energy as heat, causing thermal damage to the targeted cancer cells. We also observed that RF absorption and heat dissipation is dependent on solubility of AuNPs in the colloid, which is pH dependent. Furthermore, by modulating endolysosomal pH it is possible to prevent intracellular AuNP aggregation and enhance thermal cytotoxicity in hepatocellular cancer cells. From the Clinical EditorGold nanoparticles absorb energy from RF fields and can exert hyperthermic effects leading to cell death. Combining this known effect with antibody-based targeting of the nanoparticles, selective cancer specific hyperthermia induced cell death therapies can be designed, as demonstrated in this article.

Proteome profiling of heat shock of human primary breast epithelial cells, a dataset report

Exposure to elevated temperatures has a strong effect on cell functions, and is used in clinical practice. Hyperthermia may affect multiple regulatory mechanisms in cells. To understand better the response to hyperthermia of immortalized primary human breast epithelial cells, we performed a proteomics study of these cells cultured at 34°C or 39°C. Twenty-four proteins were shown to be differentially expressed due to hyperthermia. Analysis of these proteins showed the potential involvement of various biological processes in response to hyperthermia, e.g., cell adhesion, cell communication, and cell cycle. Transforming growth factor-β2 (TGF-β2) and heat shock protein 27 (HSP27) were found to be upregulated at 39°C. TGF-β2 was found to affect expression of HSP27, and to have a protective role in hyperthermia-induced cell death. Thus, the dataset described here of hyperthermia-related proteins in human primary breast epithelial cells predicts a number of cellular activities affected by exposure to high temperatures and provides a set of proteins for further studies.

Single-cell-based measurement of supraphysiological thermal injury in human carcinoma cells utilizing a micropatterned hydrogel chip

Hyperthermia affects certain regulatory proteins, kinases or cyclins, resulting in alternations to the cell cycle and even to apoptosis. Damage to the cell plasma membrane is a key factor in the killing of a cell by hyperthermia. Analysis at the single-cell level is necessary for understanding the fundamental mechanisms of hyperthermia-induced cell death and the generation of thermotolerance in surviving cells. Engineering approaches achieving precise control of cellular micropatterning provide the potential for investigating the mechanisms of thermal injury to cells at the single-cell level. The main purpose of this study is to fabricate a hydrogel chip with microwells for cellular patterning and to demonstrate the feasibility of measurement of supraphysiological thermal injury in human carcinoma cells (HeLa cells) at the single-cell level. To accomplish this, measurement of membrane injury by dye leakage post-thermal insult was performed and reported in this work. A hydrogel chip with microwells with different diameters was fabricated. For cell concentrations at 0.5×106 cells/mL, the occupancy of cells on the microchip with 40 µm microwells was up to 86.6%, a value far higher than that found on the 30 µm microwells (approximately 78.5%). Most microwells of 30 µm in diameter (about 70%) were occupied by a single cell. The fluorescent images showed that calcein leakage occurred when cell membranes were damaged under supraphysiological temperatures between 43 and 50°C. The normalized intensity of calcein decreased to 32% under a supraphysiological temperature of 43°C for 20 min. The intensity of calcein in cells was less than 20% under a supraphysiological temperature of 50°C. The feasibility of the single-cell-based experiment of thermal injury in the microchip with hydrogel microwells was therefore successfully demonstrated.

Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon ( Salmo salar) embryos

Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon ( Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18–20 °C for 84 h after acclimatization at 8 °C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2–2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos . Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.

Cell death and expression of heat‐shock protein Hsc70 in the hyperthermic rat brain

Stressful stimuli induce two distinct cellular reactions, the heat-shock (stress) response and programmed cell death. This study utilizes a dual procedure involving immunocytochemistry for heat-shock protein localization and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay for localization of cell death at the cellular level. Whole-body hyperthermia resulted in cell death in the adult rat thymus and testis with a robust signal seen in the testis. Distinct populations of constitutively expressed Hsc70-positive cells and TUNEL-positive cells were apparent. Cellular layers that exhibited high levels of Hsc70 were not triggered into cell death by the thermal stress. High expression of Hsc70 was observed in neuronal populations in the dentate gyrus, CA1 and CA2 region of the hippocampus and Purkinje neurons of the cerebellum. Hyperthermia-induced cell death was not observed in these neuronal cell types, suggesting that neuronal expression of constitutive Hsc70 may play roles in preprotecting neurons from stressful stimuli.