Hyperthermia-induced Apoptosis Research Articles

Ti2C3 MXene-based nanocomposite as an intelligent nanoplatform for efficient mild hyperthermia treatment

Photothermal therapy (PTT) has attracted much attention due to its less invasive, controllable and highly effective nature. However, PTT also suffers from intrinsic cancer resistance mediated by cell survival pathways. These survival pathways are regulated by a variety of proteins, among which heat shock protein (HSP) triggers thermotolerance and protects tumor cells from hyperthermia-induced apoptosis. Confronted by this challenge, we propose and validate here a novel MXene-based HSP-inhibited mild photothermal platform, which significantly enhances the sensitivity of tumor cells to heat-induced stress and thus improves the PPT efficacy. The Ti3C2@Qu nanocomposites are constructed by utilizing the high photothermal conversion ability of Ti3C2 nanosheets in combination with quercetin (Qu) as an inhibitor of HSP70. Qu molecules are loaded onto the nanoplatform in a pH-sensitive controlled release manner. The acidic environment of the tumor causes the burst-release of Qu molecules, which deplete the level of heat shock protein 70 (HSP70) in tumor cells and leave the tumor cells out from the protection of the heat-resistant survival pathway in advance, thus sensitizing the hyperthermia efficacy. The nanostructure, photothermal properties, pH-responsive controlled release, synergistic photothermal ablation of tumor cells in vitro and in vivo, and hyperthermia effect on subcellular structures of the Ti3C2@Qu nanocomposites were systematically investigated.

Cordycepin enhances hyperthermia-induced apoptosis and cell cycle arrest by modulating the MAPK pathway in human lymphoma U937 cells.

Hyperthermia induces cancer cell death. However, the cytotoxic effect of hyperthermia is not sufficient. Cordycepin can also induce apoptosis in cancer cells and enhance the antitumoral activity of irradiation. To examine cordycepin-mediated enhancement of hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and cordycepin on human leukemia U937 cells. Cell viability and apoptosis were measured using MTT assays, Hoechst 33342 staining and Annexin V/PI double staining. The distribution of the cell cycle and sub-G1 phase, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometry. The expression of related proteins was analyzed by western blotting. Combined treatment with hyperthermia and cordycepin markedly augmented apoptosis by upregulating Bax and suppressing Bcl-2, Bid and activated caspase 3 and 8 expression, and apoptosis was decreased by Z-VAD-fmk (a pan caspase inhibitor). We also found that the MMP was significantly decreased and excessive ROS generation occurred. The combination treatment also induced arrest in the G2/M phase by downregulating cyclin dependent kinase 1 (CDK1) and cyclin B1 protein expression. Furthermore, it was observed that mitogen-activated protein kinase (MAPK) pathway including ERK, JNK and p38 signals was involved in the induction of apoptosis. The phosphorylated p38 and JNK were increased and ERK phosphorylation was decreased by the combined treatment. In addition, N-acetyl-L-cysteine (NAC) significantly protected the cells by restoring ROS levels and the activity of caspase-3, inactivating the MAPK pathway. Cordycepin significantly enhanced hyperthermia-induced apoptosis and G2/M phase arrest in U937 cells. The combined treatment enhanced apoptosis through the MAPK pathway and mitochondrial dysfunction, and these effects could be rescued by NAC. We report for the first time that cordycepin can be used as a hyperthermia sensitizer to treat leukemia.

Insights into the synthesis optimization of Fe@SiO2 Core-Shell nanostructure as a highly efficient nano-heater for magnetic hyperthermia treatment

Maintaining the intact iron core, protecting the extra iron ion release, and generating biologically sufficient heat, are the critical aspects for magnetic hyperthermia treatment (MHT). Thus, the composition of silica-coated nanoscale zero-valent iron (nZVI) was optimized by applying response surface methodology (RSM) and changing the molar ratio of tetraethyl orthosilicate (TEOS) and iron(II) sulfate (FeSO4·7H2O) as the silica and iron precursors, respectively. The TEOS/FeSO4·7H2O molar ratio of 1.67 results in the maximized saturation magnetization (99.3 emu/g) and the crystalline phase of pure iron. In comparison with the previously reported studies, the synthesized core–shell nanostructures demonstrate superior heat production features. As silica coating protects the inner core from oxidation and results in more effective heat-generating seeds, nanostructures with a higher amount of silica precursors, i.e., 10.3 mm, demonstrate an efficient specific absorption rate (SAR). Moreover, the medium and higher TEOS amounts represent acceptable cytocompatibility up to 125 μg/mL and 250 μg/mL, respectively. In vitro hyperthermia evaluation depicts the cancer cell viability reduction indicating the hyperthermia-induced apoptosis. Based on the data mentioned above, we could introduce a potential successful nanoparticle for magnetic hyperthermia treatment.

Photothermal treatment by PLGA–gold nanorod–isatin nanocomplexes under near-infrared irradiation for alleviating psoriasiform hyperproliferation

Psoriasis is a chronic autoimmune skin disorder that involves keratinocyte hyperproliferation and inflammatory cell recruitment. A strategy to mitigate psoriatic lesions is to induce keratinocyte apoptosis for proliferation suppression. Herein we designed a nanoformulation capable of treating psoriasis via hyperthermia-induced apoptosis in response to near-infrared (NIR) irradiation. To this end, gold nanorods (GNRs) and isatin, which is an anti-inflammatory agent for synergizing antipsoriatic activity, were loaded into a poly (lactic-co-glycolic acid) (PLGA) matrix to form the nanocomplexes. The physicochemical and photothermal properties of the nanocomplexes were determined in terms of size, surface charge, NIR-absorbing feature, isatin release, keratinocyte uptake, and cytotoxicity. The nanocomplexes showed a spherical shape with an average size of about 180 nm. The GNR-loaded nanoparticles can efficiently convert NIR light at 0.42 W/cm2 into heat with an increased temperature of 10 °C. When combined with NIR exposure, the nanocomplexes were internalized into keratinocyte cytoplasm with an inhibition of keratinocyte viability to about 60%. Live/dead cell assay and flow cytometry confirmed that the nanocomplexes could serve as NIR-absorbers to specifically elicit keratinocyte apoptosis through caspase and poly ADP-ribose polymerase (PARP) pathways. The in vivo psoriasiform murine model indicated that the combined nanocomplexes and NIR inhibited epidermal hyperplasia and neutrophil infiltration. The overexpressed cytokines in the lesion could be recovered to normal baseline level after the photothermal management. The subcutaneous nanocomplexes remained in the skin for at least 5 days. The nanocomposites produced a negligible toxicity in the skin or liver of healthy mice. The photothermal nanosystems, as designed in this study, shed new light on the therapeutic approach against psoriasis.

Abstract No. 506 Image-guided radiofrequency hyperthermia-enhanced direct chemotherapy of hepatic tumors: the underlying biomolecular mechanisms

To evaluate the treatment effects of combined therapy using Doxorubicin and radiofrequency hyperthermia and expound the mechanism of local Chemotherapy under the heat-stress condition in vitro and in a rabbit VX2 liver tumor model Institutional Animal Care and Use Committee approved all studies. In vitro experiments VX2 cell lines cultured and were randomly assigned to four treatment groups, namely. Group A, phosphate-buffered saline (n = 6); group B, 5 μΜ Doxorubicin alone; hroup C, 42°C culture for 20 min (n = 6); and group D, 5 μΜ doxorubicin plus under 42°C culture for 20 min (n = 6). The viability and doxorubicin uptake of VX2 tumor cells line were assayed by flow cytometer and fluorescence microscopy at 24h after different treatment. Western blot was used to evaluate the Hsp70 expression. Tumor tissues were harvested from our Prestudy of rabbit liver VX2 tumor model. Fluorescence microscopy observed the distribution of doxorubicin, Hsp70 activity was analyzed by Western blot and Immunohistochemistry. P <0.05 was considered statistically significant. Radiofrequency hyperthermia-induced apoptosis and necrosis in VX2 cells in a mechanism of enhancing doxorubicin dose in cell and Hsp 70 expression. Fluorescence microscopy showed a further increase doxorubicin concentration in tumor cells and the penetration depth in tumor tissue. The western blot result showed Hsp70 expression in the combined group was significantly greater than in the control groups (mean ± SEM: 0.15 ± 0.03 vs. 0.64 ± 0.13 vs. 0.83 ± 0.10 vs. 1.28 ± 0.13, P <0.05). In the tumor tissue, immunohistochemistry staining results showed the significant increase of Hsp70-positive signaling was observed in RFH combined doxorubicin group than control group (P <0.01). Significantly increased of the tumor apoptosis and necrosis also found in the combined group with Ki-67 and TUNEL staining (P < .001). Radiofrequency hyperthermia mediate chemotherapy-induced VX2 cell killing regulated cell death mechanisms include enhance penetration of doxorubicin and HSP70 interacting at several points on apoptotic and immune signaling pathways, leads to inhibition of apoptosis of cell death at the HCC tumor ablation margin.

Protective effect of dihydromyricetin on hyperthermia-induced apoptosis in human myelomonocytic lymphoma cells.

Dihydromyricetin (DMY) is a traditional herbal medicine, with a wide range of biological activities. Extreme hyperthermia (HT) can suppress the immune system; thus, protection of the immune system is beneficial in heat-related diseases, including heatstroke. In our study, we revealed the protective effect of DMY against HT-induced apoptosis and analysed the underlying molecular mechanisms. We incubated human myelomonocytic lymphoma U937 cells at 44°C for 30min with or without DMY and followed by further incubation for 6h at 37°C. Cell viability was determined by the CCK-8 assay. DMY did not cause any cytotoxic effects in U937 cells even at high doses. HT treatment alone induced significant apoptosis, which was detected by DNA fragmentation and Annexin V/PI double staining. Mitochondrial dysfunction was identified by loss of mitochondrial membrane potential (MMP) during heat stimulation. Apoptoticrelated proteins were involved, truncated Bid and caspase-3 were upregulated, and Mcl-1 and XIAP were downregulated. We also identified the related signalling pathways, such as the MAPK and PI3K/AKT pathways. However, changes in HT were dramatically reversed when the cells were pretreated with DMY before exposure to HT. Overall, MAPKs and PI3K/AKT signalling, mitochondrial dysfunction, and caspase-mediated pathways were involved in the protective effect of DMY against HT-induced apoptosis in U937 cells, which was totally reversed by DMY pretreatment. These findings indicate a new clinical therapeutic strategy for the protection of immune cells during heatstroke.

Excessive Oxidative Stress in the Synergistic Effects of Shikonin on the Hyperthermia-Induced Apoptosis.

Hyperthermia (HT) has been used widely for cancer therapy, and the development of modern devices has made it more efficient. Shikonin (SHK) is a natural naphthoquinone derivative from a Chinese herb. Although the anticancer properties of SHK are evident, the underlying molecular mechanisms are not fully understood. In this study, the effects of combining low doses of SHK with mild HT were investigated in the U937 cell line. The cells were subjected to HT at 44°C for 10 min with or without SHK pretreatment, and parameters reflecting apoptosis, ROS generation and intracellular calcium elevation were evaluated by using DNA fragmentation, flow cytometry, and western blot analyses. SHK 0.5 µM significantly enhanced HT-induced apoptosis as indicated by DNA fragmentation and caspase-3 activation with increased generation of ROS and elevation of intracellular calcium. The combined treatment also synergistically activated proapoptotic proteins and inactivated anti-apoptotic proteins. Furthermore, the phosphorylation of JNK and PKC- δ and the dephosphorylation of ERK and AKT were the upstream effects that may have compounded the induction of apoptosis. The modulatory effects of HT and SHK were abrogated with the employment of NAC and JNK-IN-8 by inactivating the MAPK pathway and cleavage of caspase-3. Intracellular calcium was also elevated and was found to be responsible for the induction of cell death evident by the DNA fragmentation with or without the employment of BAPTA-AM. Conclusively, this study provides persuasive evidence that SHK in combination with HT is a propitious therapeutic way for augmentation of apoptosis and hence suggest a novel strategy for treating cancers.

Hyperthermia enhances the sensitivity of pancreatic cancer SW1990 cells to gemcitabine through ROS/JNK signaling.

Pancreatic cancer (PC) is a highly aggressive type of cancer. Gemcitabine (GEM) is a standard chemotherapeutic treatment of advanced PC; however, it requires improvement, and more effective therapeutic methods must be further explored. In the present study, hyperthermia combined with GEM was used on the PC cell line SW1990. The results revealed that mild hyperthermia (at 42°C) effectively increased the inhibitory effect of GEM on cell viability, as determined using an MTT assay, and increased the effect of GEM-induced apoptosis, as determined using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, in PC SW1990 cells. Additionally, it resulted in increased S-phase arrest, downregulated the expression of the anti-apoptosis protein B-cell lymphoma 2 and upregulated the expression of the pro-apoptosis protein Bcl-2-associated X protein, cleaved caspase-3 and cleaved caspase-9, as determined using a reverse transcription-quantitative polymerase chain reaction and western blot analysis. Furthermore, it was revealed that hyperthermia resulted in the rapid generation of reactive oxygen species (ROS) and substantial activation of c-Jun-N-terminal kinase (JNK). The introduction of ROS and JNK inhibitors suppressed hyperthermia-induced apoptosis in GEM-treated cells, suggesting that hyperthermia increased GEM cytotoxicity in PC SW1990 cells by inducing apoptosis via the ROS/JNK signaling pathway.

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.

Baicalin Augments Hyperthermia-Induced Apoptosis in U937 Cells and Modulates the MAPK Pathway via ROS Generation

Background/Aims: Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the flavonoid compound baicalin (BCN) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 myelomonocytic leukemia cell line. To explore any enhancing effects of BCN on hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and BCN in U937 cells. Methods: U937 cells were heat treated at 44ºC for 12 min with or without pre-treatment with BCN (10-50 µM) and then incubated for 6 h at 37 ºC with 5% CO2 and 95% air. Cell viability was analyzed by Trypan blue exclusion assay. Apoptosis was examined by DNA fragmentation, fluorescence microscopy and flow cytometry. Generation of mitochondrial trans-membrane potential (MMP), mitochondrial calcium, and reactive oxygen species (ROS) was also detected by flow cytometry. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. Results: Hyperthermia alone did not reduce cell viability or induce notable levels of apoptosis, but combined hyperthermia and BCN treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Mitochondrial transmembrane potential was significantly decreased, and generation of reactive oxygen species (ROS) and suppression of antioxidant enzymes were marked. Furthermore, with the combined treatment, the phosphorylated forms of JNK and p38 showed increased expression, whereas AKT was dephosphorylated. JNK-IN-8 (a JNK inhibitor) and NAC (a ROS scavenger) abrogated the apoptotic effects of the combined treatment, significantly protecting the cells and indicating the involvement of high ROS generation and the MAPK pathway in the underlying molecular mechanism. Conclusion: This study provides compelling evidence that hyperthermia, in combination with BCN, is a promising therapeutic strategy for enhancement of apoptosis and suggest a promising therapeutic approach for cancer.

Hyperthermia exposure induces apoptosis and inhibits proliferation in HCT116 cells by upregulating miR-34a and causing transcriptional activation of p53.

Hyperthermia, as an anticancer therapeutic strategy, presents notable advantages in conjunction with irradiation and/or chemotherapy in the treatment of cancer by promoting apoptosis and inhibiting proliferation. A number of studies have documented that hyperthermia inhibits cancer progression through transcriptional activation of p53, which promotes cell cycle arrest and apoptosis. However, the underlying molecular mechanisms of hyperthermia-regulated apoptosis and proliferation dependent on p53 remain largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis and proliferation of colorectal carcinoma (CRC) HCT116 cells, the present study assessed cell apoptosis and proliferation following exposure to hyperthermia (42°C for 2–4 h). The results indicated that, compared with the control group at 0 h, hyperthermia exposure for 2 and 4 h induced the apoptosis of HCT116 cells (P<0.05), inhibited cell proliferation by causing cell cycle arrest at G1/G0 phase (P<0.05), and significantly increased microRNA (miR)-34a expression (P<0.05), but not miR-34b, miR-34c, miR-215 and miR-504 expression. The transcriptional activity of p53 on its consensus sequence and downstream target genes, namely p21, B cell lymphoma 2-associated X protein, mouse double minute 2 homolog, p53 upregulated modulator of apoptosis and growth arrest and DNA-damage-inducible 45α, was subsequently detected. The data indicated significantly higher transcriptional activity of p53 following hyperthermia exposure for 2 and 4 h (P<0.05), and these observations were similar to the effects of transfection with miR-34a mimics in HCT116 cells. Furthermore, transfection with miR-34a antagomiR supressed hyperthermia-induced apoptosis and promoted cell cycle progression following hyperthermia exposure when compared with transfection controls (P<0.05). Collectively, these findings indicate that miR-34a may serve an important role in hyperthermia-regulated apoptosis and proliferation in HCT116 cells by influencing the transcriptional activity of p53.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in Molt-4 cells

Purpose: Transforming growth factor-β-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line.Materials and methods: Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity.Results: Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis.Conclusion: Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.

Melatonin enhances hyperthermia-induced apoptotic cell death in human leukemia cells.

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia-induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase-2, caspase-3, caspase-8, and caspase-9). Melatonin also increased hyperthermia-induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia-provoked apoptosis and potentiation by melatonin were abrogated by a broad-spectrum caspase inhibitor (z-VAD-fmk) as well as by specific inhibitors against caspase-8 or caspase-3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase-9 inhibitor or overexpressing the anti-apoptotic protein Bcl-2 (U937/Bcl-2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type-specific manner as similar results were observed in HL-60 but not in K562 or MOLT-3 cells.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in A549 cells.

KRAS mutant lung cancers have long been considered as untreatable with drugs. Transforming growth factor-β-activated kinase 1 (TAK1) appears to play an anti-apoptotic role in response to multiple stresses and has been reported to be a responsive kinase that regulates cell survival in KRAS-dependent cells. In this study, in order to find a useful approach to treat KRAS mutant lung cancer, we focused on the combined effects of 5Z-7-oxozeaenol, a TAK1 inhibitor, with hyperthermia (HT) in KRAS mutant lung cancer cell line A549. Annexin V-FITC/PI assay, cell cycle analysis, and colony formation assay revealed a significant enhancement in apoptosis induced by HT treatment, when the cells were pre-incubated with 5Z-7-oxozeaenol in a dose-dependent manner. The enhanced apoptosis by 5Z-7-oxozeaenol was accompanied by a significant increase in reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (MMP). In addition, western blot showed that 5Z-7-oxozeaenol enhanced HT-induced expressions of cleaved caspase-3, cleaved caspase-8, and HSP70 and decreased HT-induced expressions of Bcl-2, p-p38, p-JNK, and LC3. Moreover, 5Z-7-oxozeaenol pre-treatment resulted in a marked elevation of intracellular calcium level which might be associated with endoplasmic reticulum (ER) stress-related pathway. Taken together, our data provides further insights of the mechanism of action of 5Z-7-oxozeaenol and HT treatment, and their potential application as a novel approache to treat patients with KRAS mutant lung cancer.

Hyperthermia induces apoptosis by targeting Survivin in esophageal cancer

Hyperthermia is considered the fifth pillar of cancer treatment. It induces cancer cell apoptosis, however, its molecular mechanisms remain unclear. In the present study, the role of Survivin in hyperthermia-induced apoptosis in esophageal cancer was investigated. Different temperatures were used to treat EC109 esophageal cancer cells, and their viability was found to be significantly inhibited with a concomitant increase in apoptosis and necrosis. Necrosis increased in a temperature‑dependent manner, whereas peak apoptosis was reached at 43˚C. The hyperthermia-induced apoptosis was due to the inhibition of Survivin and the activation of caspase-3. Subsequently, overexpression of Survivin inhibited the activation of caspase-3 and hyperthermia-induced apoptosis, however, this inhibition was reversed in the absence of XIAP. Immunoprecipitations showed that Survivin did not directly bind to caspase-3, whereas XIAP interacted with Survivin and caspase-3. Immunohistochemistry was performed to detect the expression of Survivin in esophageal cancer patient samples. A higher expression of Survivin in esophageal cancer tissues compared to normal tissues was observed, and a high expression correlated with poor prognosis. The results indicated that hyperthermia decreases the expression of Survivin, prevents its binding to XIAP, activates caspase-3 and induces apoptosis. Due to its correlation with poor prognosis, Survivin may be a target for hyperthermia in the treatment of esophageal cancer.

Identification of proteins interacting with protein kinase C-δ in hyperthermia-induced apoptosis and thermotolerance of Tca8113 cells.

The purpose of the present study was to investigate the differential proteins that interact with protein kinase C‑δ (PKC‑δ) in hyperthermia‑induced apoptosis as well as thermotolerance in Tca8113 cells, and furthermore, to investigate the mechanisms of these processes in tumor cells. Activation of PKC‑δ was previously indicated to be involved in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells. Tca8113 cell apoptosis was induced by incubation at 43˚C for 80 min and the thermotolerant Tca8113 cells (TT‑Tca8113) were generated through a gradient temperature‑elevating method. The apoptotic rate of the cells was determined by flow cytometry, while cleavage and activation of PKC‑δ were analyzed by western blot analysis. The proteins that interacted with PKC‑δ in the Tca8113 and TT‑Tca8113 cells were identified by co‑immunoprecipitation coupled with mass spectrometry. Co‑immunoprecipitation analysis followed by electrospray ionization mass spectrometric analysis were utilized to identify the pro‑ and anti‑apoptotic proteins that interacted with PKC‑δ. Significant cell apoptosis was observed in Tca8113 cells following hyperthermia, and the apoptotic rate was significantly higher than that in the control group (P<0.05). Marked PKC‑δ cleavage fragmentation was also identified. By contrast, the apoptotic rate of the TT‑Tca8113 cells was not significantly increased following hyperthermia and no PKC‑δ cleavage fragmentation was observed. Among the proteins interacting with PKC‑δ, 39 were found to be involved in the promotion of apoptosis and 16 in the inhibition of apoptosis of Tca8113 cells; these proteins were known to be involved in the regulation of cell proliferation, apoptosis, transcription and intracellular protein transport. The results of the present study provided evidence that PKC‑δ is a crucial factor in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells and elucidated the underlying molecular basis, which may aid in the improvement of hyperthermic cancer treatments.

Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression.

Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution.

Mild thermotolerance induced at 40 °C protects cells against hyperthermia-induced pro-apoptotic changes in Bcl-2 family proteins

Purpose: Despite clinical progress, mechanisms involved in cellular responses to low and high doses of hyperthermia are not entirely clear. This study investigates the role of Bcl-2 family proteins in control of the mitochondrial pathway of apoptosis during hyperthermia at 42–43 °C and the protective effect of a low dose adaptive survival response, mild thermotolerance induced at 40 °C.Materials and methods: Levels of Bcl-2 family proteins were detected in HeLa cells by western blotting, caspase activation by spectrofluorimetry and apoptosis by chromatin condensation.Results: Hyperthermia (42–43 °C) decreased total and mitochondrial expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, while expression of pro-apoptotic proteins Bax, Bak, Puma and Noxa increased. Hyperthermia perturbed the equilibrium between these anti- and pro-apoptotic Bcl-2 family proteins in favour of pro-apoptotic conditions. Hyperthermia also caused activation of caspases-9 and -3, and chromatin condensation. Disruption of the balance between Bcl-2 family proteins was reversed in thermotolerant (40 °C) cells, thus favouring cell survival. Bcl-2/Bcl-xL inhibitor ABT-737 sensitised cells to apoptosis, which indicates that Bcl-2 family proteins play a role in hyperthermia-induced apoptosis. The adaptive response of mild thermotolerance (40 °C) was still able to protect cells against hyperthermia (42–43 °C) when Bcl-2/Bcl-xL were inhibited.Conclusions: These results improve knowledge about the role of Bcl-2 family proteins in cellular apoptotic responses to hyperthermia (42–43 °C), as well as the adaptive survival response induced by exposure to mild stresses, such as a fever temperature (40 °C). This study could provide rationale to explore the manipulation of Bcl-2 family proteins for increasing tumour sensitivity to hyperthermia.

The protective effect of rosmarinic acid on hyperthermia-induced C2C12 muscle cells damage.

High temperature will cause animal tissues or cells damage. Rosmarinic acid (RA) is a good antioxidant and health care product, but the roles of RA in muscle cells damage and the mechanisms which caused by high temperature is still unknown. In this study, the roles of RA on hyperthermia-induced apoptosis and damage of C2C12 muscle cells were investigated. C2C12 cells were cultured in medium with different concentration (0, 25, 50, 100 µM) RA and treated in 42 °C high temperature to induce cellular apoptosis and damage. Then, these cells were analyzed effect of different dose of RA on cells apoptosis and damage. The results indicated that RA has protective effect on heat-stress induced cellular damage, and the cells have the higher cell viability at the dose of 50 µM RA by MTT assay. Hochest33342/PI double staining showed that the cellular apoptosis of C2C12 cells were decreased in the presence of selected 50 µM RA. Malondialdehyde formation and reactive oxygen species levels were also decreased significantly, but cellular superoxide dismutase activity was increased significantly in the presence of RA even in the condition of 42 °C. Meanwhile, Caspase-3 mRNA expression, Caspase-3 activity, and Bax/Bcl-2 ratio were reduced significantly, but the mRNA expression of Hsp72 was increased significantly in those hyperthermia-induced C2C12 cells in the presence of 50 µM RA. Taken together, the results at least discovered that RA has protective effects on hyperthermia-induced cellular apoptosis and damage of muscle cells by change the expression of stress-genes and increasing intracellular antioxidant capability.

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.