Effect of hyperthermia on the antitumor actions of interferons.

Hyperthermia treatment has been shown to enhance the in vitro antiproliferative effects of IFN-alpha, IFN-beta, and IFN-gamma, with IFN-gamma being more strongly enhanced than IFN-alpha. The comparative effects of hyperthermia on the in vivo antitumor activities of IFN-alpha and IFN-gamma were evaluated in the murine system using both subcutaneous and intraperitoneal B16 melanoma tumor model systems. Heat-induced whole body hyperthermia, resulting in a 2 degree C rise in body temperature, was administered by incubating the mice for 8 hours in a dry incubator at 37.1 degrees C. Whole body hyperthermia was found to enhance the antitumor activity of IFN-alpha by approximately 1.0 fold and 1.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented an additive effect of hyperthermia and IFN-alpha. Hyperthermia was found to enhance the antitumor activity of IFN-gamma by approximately 2.9 fold and 2.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented a synergistic effect of hyperthermia and IFN-gamma. The results of this in vivo study confirm and extend the in vitro observation that hyperthermia more strongly enhances the antitumor action of IFN-gamma than IFN-alpha. These results may have clinical importance because they suggest that hyperthermia may be used in combination with IFN-gamma to provide a synergistically enhanced antitumor action.     

Differential effects of hyperthermia on human leukocyte production of interferon-alpha and interferon-gamma

Hyperthermia is being used clinically in the treatment of neoplasms. However, there are insufficient data regarding effects of hyperthermia on leukocyte functions potentially important in antitumor immunity. In order to provide such data, human mononuclear leukocytes were exposed to moderate (40.7 degrees C) and marked (42.7 degrees C) hyperthermia for 2 hr. Leukocyte viability, measured by dye exclusion, was not altered by such exposures. Exposure of the cells to moderate hyperthermia did not alter leukocyte production of interferon-alpha in response to influenza virus or interferon-gamma in response to the mitogen phytohemagglutinin. Exposure of the cells to marked hyperthermia significantly depressed production of interferon-alpha. In contrast, production of interferon-gamma was not altered by exposure of the leukocytes to marked hyperthermia. Many studies support a role for interferons (alpha as well as gamma) in antitumor immunity. The current and other data suggest that marked hyperthermia in cancer therapy should be applied locally whenever possible, rather than to the whole body, in order to limit adverse effects on immunity. The data suggest further that interferon-gamma may be a heat shock (stress) protein for human leukocytes.     

Sublethal hyperthermia enhances anticancer activity of doxorubicin in chronically hypoxic HepG2 cells through ROS-dependent mechanism

Thermal ablation in combination with transarterial chemoembolization (TACE) has been reported to exert a more powerful antitumor effect than thermal ablation alone in hepatocellular carcinoma patients. However, the underlying mechanisms remain unclear. The purpose of the present study was to evaluate whether sublethal hyperthermia encountered in the periablation zone during thermal ablation enhances the anticancer activity of doxorubicin in chronically hypoxic (encountered in the tumor area after TACE) liver cancer cells and to explore the underlying mechanisms. In the present study, HepG2 cells precultured under chronic hypoxic conditions (1% oxygen) were treated in a 42°C water bath for 15 or 30 min, followed by incubation with doxorubicin. Assays were then performed to determine intracellular uptake of doxorubicin, cell viability, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and total antioxidant capacity. The results confirmed that sublethal hyperthermia enhanced the intracellular uptake of doxorubicin into hypoxic HepG2 cells. Hyperthermia combined with doxorubicin led to a greater inhibition of cell viability and increased apoptosis in hypoxic HepG2 cells as compared with hyperthermia or doxorubicin alone. In addition, the combination induced apoptosis by increasing ROS and causing disruption of MMP. Pretreatment with the ROS scavenger N-acetyl cysteine significantly inhibited the apoptotic response, suggesting that cell death is ROS-dependent. These findings suggested that sublethal hyperthermia enhances the anticancer activity of doxorubicin in hypoxic HepG2 cells via a ROS-dependent mechanism.     

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133⁺/CD44⁺ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133⁺/CD44⁺ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.     

Effects of hyperthermia on the in vitro antiproliferative activities of HuIFN-alpha, HuIFN-beta, and rHuIFN-gamma employed separately and in combination

The relative enhancing effects of hyperthermia on the three types of interferon were evaluated in cloning studies for three human cell lines: G-361 malignant melanoma cells, WISH ammion cells, and AGS stomach adenocarcinoma cells. Hyperthermia enhanced the antiproliferative activity of rHuIFN-gamma against each of the three cell lines and the levels of enhancement by hyperthermia were seen to increase with increasing concentrations of rHuIFN-gamma. The maximum observed levels of enhancement of rHuIFN-gamma activity by hyperthermia varied from cell line to cell line. However, when the relative sensitivities of the cell lines to rHuIFN-gamma were taken into account, the levels of enhancement of rHuIFN-gamma antiproliferative activity by hyperthermia were seen to be similar for each of the cell lines, indicating that hyperthermia consistently enhanced rHuIFN-gamma antiproliferative activity. Hyperthermia did not consistently enhance the antiproliferative activities of HuIFN-alpha and HuIFN-beta. Further studies indicated that hyperthermia enhanced by approximately 6-fold the antiproliferative effects of combinations of rHuIFN-gamma with HuIFN-alpha and HuIFN-beta. The results support the possibility that a combination treatment protocol of hyperthermia and interferon administration (particularly HuIFN-gamma or combinations of HuIFN-gamma with HuIFN-alpha or HuIFN-beta) may provide an enhanced antitumor effect in man.     

Antitumor effects of combined therapy of recombinant heat shock protein 70 and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma

Heat shock proteins (HSPs) are recognized as significant participants in cancer immunity. We previously reported that HSP70 expression following hyperthermia using magnetic nanoparticles induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by administration of recombinant HSP70 protein into the tumor in situ. Hyperthermia was conducted using our original magnetite cationic liposomes (MCLs), which have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs and recombinant mouse HSP70 (rmHSP70) were injected into melanoma nodules in C57BL/6 mice, which were subjected to AMF for 30 min. Temperature within the tumor reached 43°C and was maintained by controlling the magnetic field intensity. The combined treatment strongly inhibited tumor growth over a 30-day period and complete regression of tumors was observed in 20% (2/10) of mice. It was also found that systemic antitumor immunity was induced in the cured mice. This study suggests that novel combined therapy using exogenous HSP70 and hyperthermia has great potential in cancer treatment.     

Combinatorial effects of radiofrequency hyperthermia and radiotherapy in the presence of magneto-plasmonic nanoparticles on MCF-7 breast cancer cells

Here, the effects of combinatorial cancer therapy including radiotherapy (RT) and radiofrequency (RF) hyperthermia in the presence of gold-coated iron oxide nanoparticles (Au@IONPs), as a thermo-radio-sensitizer, are reported. The level of cell death and the ratio of Bax/Bcl2 genes, involved in the pathway of apoptosis, were measured to evaluate the synergistic effect of Au@IONPs-mediated RF hyperthermia and RT. MCF-7 human breast adenocarcinoma cells were treated with different concentrations of Au@IONPs. After incubation with NPs, the cells were exposed to RF waves (13.56 MHz; 100 W; 15 min). At the same time, thermometry was performed with an infrared (IR) camera. Then, the cells were exposed to 6 MV X-ray at various doses of 2 and 4 Gy. MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) assay was performed to evaluate cell viability and quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression ratio of Bax/Bcl2. Cellular uptake of nanoparticles was confirmed qualitatively and quantitatively. The results obtained from MTT assay and qRT-PCR studies showed that NPs and RF hyperthermia had no significant effect when applied separately, while their combination had synergistic effects on cell viability percentage and the level of apoptosis induction. A synergistic effect was also observed when the cancer cells were treated with a combination of NPs, RF hyperthermia, and RT. On the basis of the obtained results, it may be concluded that the use of magneto-plasmonic NPs in the process of hyperthermia and RT of cancer holds a great promise to develop a new combinatorial cancer therapy strategy.     

Tumor cell apoptosis,lymphocyte recruitment and tumor vascular changes are induced by low temperature,long duration (fever-like) whole body hyperthermia

A single treatment of low-temperature, long-duration, whole-body hyperthermia of either severe combined immunodeficient (SCID) mice bearing human breast tumor xenografts or Balb/c mice bearing syngeneic tumors for 6–8 hr can cause a temporary reduction of tumor volume and/or a growth delay. In both animal model systems, this inhibition is correlated with the appearance of large numbers of apoptotic tumor cells. Because this type of mild heat exposure, comparable to a common fever, is not itself directly cytotoxic, other explanations for the observed tumor cell death were considered. Our data support the hypothesis that this hyperthermia protocol stimulates some component(s) of the immune response, which results in increased antitumor activity. In support of this hypothesis, increased numbers of lymphocyte-like cells, macrophages, and granulocytes are observed in the tumor vasculature and in the tumor stroma immediately following this mild hyperthermia exposure. In Balb/c mice, an infiltrate persists in the tumor for at least 2 weeks. Using the SCID mouse/human tumor system, we found that both host natural killer (NK) cells and injected human NK cells were increased at the site of tumor following hyperthermia treatment. Experiments using anti-asialo-GM1 antibodies indicate that the tumor cell apoptosis seen in the SCID mouse appears to be due largely to the activity of NK cells, although additional roles for other immunoeffector cells and cytokines appear likely in the immunologically complete Balb/c model. Another interrelated hypothesis is that immunoeffector cells may have greater access to the interior of the tumor because we have observed that this treatment causes an obvious expansion in the diameter of blood vessels within the tumor and an increase in nucleated blood cells within the vessels, which persists as long as 2 weeks after treatment. Further study of the mechanisms by which mild hyperthermia exerts antitumor activity could result in this treatment protocol being used as an effective, nontoxic adjuvant to immunotherapy and/or other cancer therapies. J. Cell. Physiol. 177:137–147, 1998. © 1998 Wiley-Liss, Inc.     

Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of “heat-controlled necrosis” with heat shock protein expression

Heat shock proteins (HSPs) are highly conserved proteins whose syntheses are induced by a variety of stresses, including heat stress. Since the expression of HSPs, including HSP70, protects cells from heat-induced apoptosis, HSP expression has been considered to be a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96), in immune reactions. If HSP expression induced by hyperthermia is involved in tumor immunity, novel cancer immunotherapy based on this novel concept can be developed. In such a strategy, a tumor-specific hyperthermia system, which can heat the local tumor region to the intended temperature without damaging normal tissue, would be highly advantageous. To achieve tumor-specific hyperthermia, we have developed an intracellular hyperthermia system using magnetite nanoparticles. This novel hyperthermia system can induce necrotic cell death via HSP expression, which induces antitumor immunity. In the present article, cancer immunology and immunotherapy based on hyperthermia, and HSP expression are reviewed and discussed. This article forms part of the Symposium in Writing "Thermal stress-related modulation of tumor cell physiology and immune responses", edited by Elfriede Noessner.     

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.     

Synthesis and antitumor activity of 1- or 3-(alpha-hetero substituted)alkyl-5-fluorouracil derivatives

Two classes of 5-fluorouracil (5-FU) derivatives were prepared from 1,3-bis(hydroxymethyl)-5-fluorouracil (1). The first group was obtained by the direct esterification of 1 with various acids. The second one was derived by the nucleophilic substitution of N-(chloromethyl)-5-FUs, which were easily prepared by halogenation of 1, with hetero nucleophiles. The antitumor activities of the prepared compounds were examined against Leukemia L1210 and the results were compared with that of 1-hexylcarbamoyl-5-fluorouracil (HCFU, Carmofur).     

Effect of hyperthermia in combination with TRAIL on the JNK‐Bim signal transduction pathway and growth of xenograft tumors

Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). In this study, we used human colorectal cancer CX‐1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim_EL activity as well as PARP cleavage. We have also used our CX‐1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42°C, we were able to show a delayed onset of tumor growth in our xenograft model. J. Cell. Biochem. 110: 1073–1081, 2010. Published 2010 Wiley‐Liss, Inc.     

DC-CIK治疗大肠癌的研究进展

大肠癌是消化道常见的恶性肿瘤之一,发病率和死亡率均较高。过继免疫治疗是当今肿瘤治疗的热点,已逐步成为一些肿瘤的首选治疗方法。树突状细胞(DC)是目前已知功能最强大的抗原呈递细胞,具有呈递肿瘤抗原和抵制肿瘤细胞免疫逃逸及刺激T淋巴细胞产生免疫应答的作用。细胞因子诱导的杀伤细胞(CIK)由多种细胞因子诱导而成,具有T淋巴细胞及NK细胞抗肿瘤作用的特点。DC和CIK细胞有效结合可以同时促进DC细胞的增殖和免疫功能及加强CIK细胞的抗肿瘤作用。本文就近年来国内外应用DC-CIK治疗大肠癌的研究进展进行综述。     

Effect of hyperthermia and chemotherapeutic agents on TRAIL-induced cell death in human colon cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.     

Cancer hyperthermia using magnetic nanoparticles

Magnetic-nanoparticle-mediated intracellular hyperthermia has the potential to achieve localized tumor heating without any side effects. The technique consists of targeting magnetic nanoparticles to tumor tissue followed by application of an external alternating magnetic field that induces heat through Néel relaxation loss of the magnetic nanoparticles. The temperature in tumor tissue is increased to above 43°C, which causes necrosis of cancer cells, but does not damage surrounding normal tissue. Among magnetic nanoparticles available, magnetite has been extensively studied. Recent years have seen remarkable advances in magnetite-nanoparticle-mediated hyperthermia; both functional magnetite nanoparticles and alternating-magnetic-field generators have been developed. In addition to the expected tumor cell death, hyperthermia treatment has also induced unexpected biological responses, such as tumor-specific immune responses as a result of heat-shock protein expression. These results suggest that hyperthermia is able to kill not only local tumors exposed to heat treatment, but also tumors at distant sites, including metastatic cancer cells. Currently, several research centers have begun clinical trials with promising results, suggesting that the time may have come for clinical applications. This review describes recent advances in magnetite nanoparticle-mediated hyperthermia.     

Hyperthermic treatment of DMBA-induced rat mammary cancer using magnetic nanoparticles

Background

We have developed magnetite cationic liposomes (MCLs) and applied them as a mediator of local hyperthermia. MCLs can generate heat under an alternating magnetic field (AMF). In this study, the in vivo effect of hyperthermia mediated by MCLs was examined using 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary cancer as a spontaneous cancer model.

Method

MCLs were injected into the mammary cancer and then subjected to an AMF.

Results

Four rats in 20 developed mammary tumors at more than 1 site in the body. The first-developed tumor in each of these 4 rats was selected and heated to over 43°C following administration of MCLs by an infusion pump. After a series of 3 hyperthermia treatments, treated tumors in 3 of the 4 rats were well controlled over a 30-day observation period. One of the 4 rats exhibited regrowth after 2 weeks. In this rat, there were 3 sites of tumor regrowth. Two of these regrowths were reduced in volume and regressed completely after 31 days, although the remaining one grew rapidly. These results indicated hyperthermia-induced immunological antitumor activity mediated by the MCLs.

Conclusion

Our results suggest that hyperthermic treatment using MCLs is effective in a spontaneous cancer model.

     

Effects of hyperthermia on heat shock protein expression, alkaline phosphatase activity and proliferation in human osteosarcoma cells

Hyperthermia can be used as a possible adjuvant therapy in treatment of cancer patients. In this study, the direct effect of hyperthermia on osteosarcoma derived cell lines HOS85, MG-63 and SaOS-2 was investigated. Heat shock at 42 degrees C inhibited proliferation significantly in all three cell lines tested. Furthermore a sub-lethal heat shock (42 degrees C, 1 h) decreases alkaline phosphatase activity, the absolute marker for osteoblast-like cells, in all of the three cell lines. Hsp70 was expressed constitutively and was found to be upregulated in a time-dependent manner; by up to 150% in Western blot analysis. The results of this study indicate that heat shock has an inhibitory effect on human osteosarcoma cells. These data suggest that hyperthermia has an anti-tumour effect on cancers of the bone and might, therefore, become an adjuvant treatment option.     

Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia

Heat shock proteins are recognized as significant participants in immune reactions. In this study, we have demonstrated that the cell surface presentation of MHC class I antigen was increased in tandem with increased heat shock protein 70 (HSP70) expression and the immunogenicity of rat T-9 glioma cells was enhanced by hyperthermia. T-9 cells showed growth inhibition for 24 h after the heat treatment at 43 degrees C for 1 h in vitro, but then resumed a normal growth rate. HSP70 expression reached a maximum at 24 h after heating. Flow cytometric analysis revealed a significant increase in MHC class I antigen on the surface of the heated cells. The augmentation of MHC class I surface expression started 24 h after heating and reached a maximum 48 h after heating. The expression of other immunologic mediators, such as intracellular adhesion molecule-1 (ICAM-1) and MHC class II antigens, did not increase. In an in vivo experiment using immunocompetent syngeneic rats (F344), growth of the heated T-9 cells, with augmentation of MHC class I antigen surface expression, was significantly inhibited, while the cells grew progressively in nude rats (F344/N Jcl-rnu). Furthermore, compared with lymphocytes from non-immunized (PBS only injection) rats or rats injected with non-heated T-9 cells, the splenic lymphocytes of the rats in which the heated T-9 cells were injected displayed specific cytotoxicity against T-9 cells. These results suggest that HSP70 is an important modulator of tumor cell immunogenicity, and that hyperthermic treatment of tumor cells can induce the host antitumor immunity via the expression of HSP70. These results may benefit further efforts on developing novel cancer immunotherapies based on hyperthermia.     

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.