TAK1 promotes cell survival by TNFAIP3 and IL-8 dependent and NF-κB independent pathway in HeLa cells exposed to heat stress.

PURPOSE: Transforming growth factor-ß-activated kinase 1 (TAK1) plays a role in inhibiting apoptosis in response to multiple stresses. In the present study, we investigated the role of TAK1 in cell death induced by heat stress (HS). MATERIALS AND METHODS: TAK1 knockdown HeLa cells and their parental cells were exposed to HS at 44 °C for 15, 30, 45 min followed by colony formation assay. Heat shock proteins (HSPs) induction, NF-κB phosphorylation, and caspase-3 cleavage were estimated by western blotting using specific antibodies. Global gene expression analysis was performed using the GeneChip® system. The anti-apoptotic roles of the identified genes were elucidated using small interfering RNAs targeting those genes. RESULTS: Heat sensitivity estimated by colony formation assay and caspase-3 cleavage increased in TAK1 knockdown cells. This sensitisation was not due to alterations in HSP induction or NF-κB phosphorylation as the expression levels of these proteins did not differ significantly between the TAK1 knockdown and the parent cells after HS exposure. The GeneChip® analysis revealed differences in gene expression between both cell variants after HS exposure and defined the genetic network associated with cell death. TNF-α interacting protein 3 (TNFAIP3) and Interleukin 8 (IL-8) are two of the identified genes. RNA interference against these genes increased the cleavage of caspase-3 and cell death after HS exposure. CONCLUSION: Our findings reveal the role of TAK1 in thermoresistance and show that the mediation is independent of NF-κB phosphorylation but is dependent on TNFAIP3 and IL-8 induction.

Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification.

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

Mild hyperthermia prior to electroporation increases transfection efficiency in HCT 116, HeLa S3 and SGC 7901 cells.

The change in transfection efficiency of electroporation by the combined treatment with mild preheating (40 degrees C for 30 min) was investigated. HCT 116, HeLa S3 and SGC 7901 cells were treated with electroporation in medium containing pBKCMV-Luc plasmid with or without preheating. After 24 h, luciferase activity was increased by 36, 28 and 77%; luciferase mRNA transcription was increased by 45, 50 and 68%; and fluorescein isothiocyanate-dextran accumulation was increased by 9, 35 and 15% in preheated groups, respectively. These results demonstrate that the transfection efficiency was enhanced by mild preheating. The mechanism partially involves increased macromolecular particle accumulation.

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole.

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca(2+)](i), and a decrease in the Hsp70 expression levels.

Design, synthesis, and biological evaluation of artificial macrosphelides in the search for new apoptosis-inducing agents.

Various artificial macrosphelides were designed and synthesized, including ring-enlarged analogues and epothilone-hybrid compounds. Syntheses were accomplished in an efficient manner by using a ring-closing metathesis (RCM) strategy in a key macrocyclization step. Biological evaluation of these new macrosphelide-based derivatives revealed that several epothilone hybrids, in which a thiazole-containing side chain was incorporated, exhibited potent apoptosis-inducing activity toward human lymphoma cells. These activities were considerably enhanced relative to those of natural macrosphelide compounds. Structure-activity relationship studies revealed that the "ene-dicarbonyl" substructure is apparently essential for bioactivity.

Enhancement of hyperthermia-induced apoptosis by sanazole in human lymphoma U937 cells.

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. The aim of the present study was to investigate whether sanazole enhances apoptosis induced by hyperthermia at 44 degrees C for 20 min in human lymphoma U937 cells. Sanazole alone induced continuous increase in the intracellular superoxide generation in a time-dependent manner and transient increase in the peroxide formation, which further were enhanced at 1 hour after HT treatment. Moreover, when the cells were treated first with 10 mM sanazole for 40 min, exposed to HT at 44 degrees C for 20 min and the cells were further treated with the drug at 37 degrees C for 6 h, a significant enhancement of HT-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Studying the apoptotic pathways involved in this enhancement, we found that loss of the mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, and activation of caspase-3 and caspase-8 was enhanced significantly in the U937 cells after the combined treatment. Moreover, this combination enhanced activation of Bid, and down regulation of Hsp70. In addition, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), and externalization of Fas were observed immediately after sanazole and HT treatment. Our data indicate that sanazole can enhance the hyperthermia induced-apoptosis through the Fas-caspase-8- and [Ca(2+)](i)-dependent apoptotic pathways. In addition, the down regulation of Hsp70 contributed to this enhancement.

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of benzocycloalkene compounds.

The aim of this study was to examine whether, a new synthesized class of benzocycloalkene derivatives (BCs), enhances apoptosis induced by hyperthermia. The combined effects of hyperthermia (44 degrees C, 20 min) and BCs on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (BC1 approximately 9), the combined treatment of 10 muM BC2 or BC4 and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia. And enhancement of hyperthermia-induced apoptosis by BC2 or BC4 in a dose-dependent manner was observed. When the cells were treated first with BC2 or BC4 at a nontoxic concentration of 20 muM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Flow cytometry revealed an increase of intracellular superoxide due to BC2 or BC4, which was further increased when hyperthermia was combined. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by BC2 or BC4. An increase in the intracellular Ca2+ concentration [Ca2+](i), externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide generated by BC2 or BC4 is involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca2+](i)-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.

Enhancement of sodium butyrate-induced cell death and apoptosis by X-irradiation in the human colorectal cancer cell line HCT 116.

In this study, we aimed at evaluating the possible enhancing effect exerted by the combined use of sodium butyrate (SB) and X-rays on eradicating the human colorectal cancer cell line HCT 116 containing wild-type p53. We assessed the effect of this combination on the molecular pathways leading to cell death. HCT 116 cells were subjected to SB (1 mM) treatment followed by X-irradiation (5 Gy), and the effects on cell death, cell proliferation and cell cycle were examined. We also analyzed the apoptosis-indicating protein expression, mitochondrial membrane potential and intracellular superoxide formation. Treatment with SB alone significantly induced cell cycle arrest and apoptosis, whereas X-irradiation showed no effect on cell death despite its ability to block cell proliferation. Growth arrest and cell death were enhanced in the combined treatment groups. A marked reduction in the growth rate of the combined-treatment group was observed compared to that of the single-treatment groups. The apoptotic mitochondrial pathway was significantly enhanced with the combined use of the two agents. It was observed to be involved in the increased expression levels of p53 and p21, as well as in the release of cytochrome c and the alteration of the balance of anti- and pro-apoptotic Bcl-2 family proteins. Enhanced superoxide formation was also observed. However, the death receptor pathway was found to play no role in this phenomenon. These results suggest that X-irradiation promotes cell killing in synergy with SB treatment. Thus, the combined treatment led to a mutual potentiation of the killing effects of each agent.

Enhancement of sodium butyrate-induced cell death by hyperthermia in HCT 116 human colorectal cancer cells.

AIM: The possible enhancing effect of the combined use of sodium butyrate (SB) and hyperthermia to kill HCT 116 cells was evaluated. MATERIALS AND METHODS: HCT 116 cells were subjected to SB (1 mM) treatment followed by hyperthermia (44 degrees C 60 min) and the effects on cell death, cell proliferation and the cell cycle were examined. Apoptosis-indicating protein expressions and intracellular superoxide formation were also analysed. RESULTS: A marked reduction in the growth rate of the combined-treatment group was observed compared to those of the single-treatment groups. This involved the increased expression of p53 and p21, the alteration of the balance of anti- and proapoptotic Bcl-2 family proteins and enhanced superoxide formation. However, the death receptor pathway played no role. CONCLUSION: Hyperthermia synergistically promoted cell death induced by SB. Thus, the combined treatment led to mutual potentiation of the killing effects of each agent.

Study on like-stem characteristics of tumor sphere cells in human gastric cancer line HGC-27.

Stem-like cancer cells are called cancer stem cells (CSCs) or tumor stem cells (TSCs). Methods for sorting CSCs are mainly based on the marker (CD133+/CD44+) or side population cells. However, CD133+/CD44+ cells or side population cells are very rare or even undetectable. In the present study, the tumor sphere of human gastric cancer (HGC) cell line HGC-27 was used for CSCs enrichment, and stem-like characteristics were verified by Hoechst 33342 staining technology, cell growth rate assays, sphere differentiation assay, clone formation, chemotherapy resistance study and tumor formation in an animal model. Our results demonstrated that the tumor sphere cells of HGC-27 cell line could be used to enrich CSCs, which may contribute to human gastric cancer stem cell biology research.

Expression of Fas ligand and caspase-3 contributes to formation of immune escape in gastric cancer.

AIM: To study the role of Fas ligand (FasL) and Caspase-3 expression in carcinogenesis and progression of gastric cancer and molecular mechanisms of relevant immune escape. METHODS: FasL and Caspase-3 expression was studied in adjacent epithelial cells, cancer cells and lymphocytes of primary foci, and cancer cells of metastatic foci from 113 cases of gastric cancer by streptavidin-biotin-peroxidase (S-P) immunohistochemistry. Expression of both proteins in cancer cells of primary foci was compared with clinicopathological features of gastric cancer. The relationship between FasL expression in cancer cells and Caspase-3 expression in cancer cells or infiltrating lymphocytes of primary foci was investigated. RESULTS: Cancer cells of primary foci expressed FasL in 53.98 % (61/113) of gastric cancers, more than their adjacent epithelial cells (34.51 %, 39/113) (P=0.003, chi(2)=8.681), while the expression of Caspase-3 in cancer cells of primary foci was detected in 32.74 % (37/113) of gastric cancers, less than in the adjacent epithelial cells (50.44 %, 57/113) (P=0.007, chi(2)=7.286). Infiltrating lymphocytes of the primary foci showed positive immunoreactivity to Caspase-3 in 70.80 % (80/113) of gastric cancers, more than their corresponding adjacent epithelial cells (P=0.001, chi(2)=10.635) or cancer cells of primary foci (P=0.000, chi(2)=32.767). FasL was less expressed in cancer cells of metastases (51.16 %, 22/43) than in those of the corresponding primary foci (81.58 %, 31/38) (P=0.003, chi(2)=9.907). Conversely, Caspase-3 was more expressed in cancer cells of metastases (58.14 %, 25/43) than in those of the corresponding primary foci (34.21 %, 13/38) (P=0.031, chi(2)=4.638). FasL expression was significantly correlated with tumor size (P=0.035,rs=0.276), invasive depth (P=0.039, rs=0.195), metastasis (P=0.039, rs=0.195), differentiation (P=0.015, rs=0.228) and Lauren's classification (P=0.038, rs=0.196), but not with age or gender of patients, growth pattern or TNM staging of gastric cancer (P>0.05). In contrast, Caspase-3 expression showed no correlation with any clinicopathological parameters described above in cancer cells of the primary foci (P>0.05). Interestingly, FasL expression in primary gastric cancer cells paralleled to Caspase-3 expression in infiltrating lymphocytes of the primary foci (P=0.016, chi(2)=5.825). CONCLUSION: Up-regulated expression of FasL and down-regulated expression of Caspase-3 in cancer cells of primary foci play an important role in gastric carcinogenesis. As an effective marker to reveal the biological behaviors, FasL is implicated in differentiation, growth, invasion and metastasis of gastric cancer by inducing apoptosis of infiltrating lymphocytes. Chemical substances derived from the primary foci and metastatic microenvironment can inhibit the growth of metastatic cells by enhancing Caspase-3 expression and diminishing FasL expression.

TGF-ß-activated kinase 1 promotes cell cycle arrest and cell survival of X-ray irradiated HeLa cells dependent on p21 induction but independent of NF-κB, p38 MAPK and ERK phosphorylations.

Transforming growth factor-ß-activated kinase 1 (TAK1) appears to play a role in inhibiting apoptotic death in response to multiple stresses. To assess the role of TAK1 in X-ray induced apoptosis and cell death, we irradiated parental and siRNA-TAK1-knockdown HeLa cells. Changes in gene expression levels with and without TAK1-knockdown were also examined after irradiation to elucidate the molecular mechanisms involved. After X-ray irradiation, cell death estimated by the colony formation assay increased in the TAK1-knockdown cells. Apoptosis induction, determined by caspase-3 cleavage, suggested that the increased radiosensitivity of the TAK1-knockdown cells could be partially explained by the induction of apoptosis. However, cell cycle analysis revealed that the percentage of irradiated cells in the G(2)/M-phase decreased, and those in the S- and SubG(1)-phases increased due to TAK1 depletion, suggesting that the loss of cell cycle checkpoint regulation may also be involved in the observed increased radiosensitivity. Interestingly, significant differences in the induction of NF-κB, p38 MAPK and ERK phosphorylation, the major downstream molecules of TAK1, were not observed in TAK1 knockdown cells compared to their parental control cells after irradiation. Instead, global gene expression analysis revealed differentially expressed genes after irradiation that bioinformatics analysis suggested are associated with cell cycle regulatory networks. In particular, CDKN1A (coding p21(WAF1)), which plays a central role in the identified network, was up-regulated in control cells but not in TAK1 knockdown cells after X-ray irradiation. Si-RNA knockdown of p21 decreased the percentage of cells in the G(2)/M phase and increased the percentage of cells in the S- and SubG(1)-phases after X-ray irradiation in a similar manner as TAK-1 knockdown. Taken together, these findings suggest that the role of TAK1 in cell death, cell cycle regulation and apoptosis after X irradiation is independent of NF-κB, p38 MAPK, and ERK phosphorylation, and dependent, in part, on p21 induction.

Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells.

Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSox(TM) Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation.

Parafibromin expression is an independent prognostic factor for colorectal carcinomas.

Parafibromin is a protein encoded by hyperparathyroidism 2, and its down-regulated expression is involved in the pathogenesis of parathyroid, breast, and gastric carcinomas. This study aimed to clarify the roles of parafibromin expression in tumorigenesis, progression, and prognosis of colorectal carcinomas. Parafibromin-expressing plasmid was transfected into DLD-1 cells with the phenotypes, and related molecules were examined. Parafibromin expression was examined in colorectal samples by immunohistochemistry, in situ hybridization, Western blot, or reverse transcription polymerase chain reaction. It was found that parafibromin overexpression could cause G1 arrest and enhance differentiation of DLD-1 cells. There was a high expression of p21, p27, and cyclin E, but low expression of cyclin D1 messenger RNA, phospho-cdc2, and phospho-cdc25c proteins. Parafibromin could inhibit c-myc messenger RNA expression by binding to c-myc promoter. Expression levels of nuclear parafibromin and parafibromin messenger RNA were decreased from colorectal nonneoplastic mucosa and adenomas to carcinomas (P < .05). Immunohistochemically, parafibromin expression was inversely correlated with tumor size, depth of invasion, lymph node metastasis, clinicopathologic staging, and poor prognosis of carcinomas (P < .05). It was suggested that parafibromin overexpression might suppress cell cycle progression and promote differentiation of DLD-1 cells. Aberrant parafibromin expression possibly contributes to the pathogenesis, growth, invasion, and metastasis of colorectal carcinomas and could be regarded as an independent factor to indicate a favorable prognosis for patients with colorectal carcinomas.

Nuclear or cytoplasmic localization of Bag-1 distinctly correlates with pathologic behavior and outcome of gastric carcinomas.

Bag-1 is an antiapoptotic protein with its altered expression and localization in malignancies. To clarify the role of Bag-1 in gastric carcinogenesis, its expression was examined by immunohistochemistry and in situ hybridization on a tissue microarray containing gastric carcinomas, adjacent nonneoplastic mucosa (NNM), adenomas, intestinal metaplasia (IM), or gastritis. Gastric carcinoma tissue and cell lines were studied for Bag-1 expression by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that Bag-1 proteins were differentially expressed in the nucleus or cytosol of MKN28, AGS, MKN45, KATO-III, or HGC-27 cell lines, despite similar levels of messenger RNA (mRNA) expression. The Bag-1 mRNA overexpression was detectable in 73.3% of 15 gastric carcinomas without significant difference in its encoding products' levels. The nuclear Bag-1 expression gradually decreased from gastritis, IM, adenoma to carcinoma (P < .05), and negatively correlated with lymphatic invasion or lymph node metastasis, cytoplasmic Bag-1 expression, negative parafibromin expression, and poor prognosis (P < .05). Cytoplasmic Bag-1 was weakly immunoreactive in carcinomas, compared with gastritis (P < .05), and positively associated with invasive depth and poor prognosis of the carcinoma (P < .05). The positive rate of Bag-1 mRNA expression was higher in adjacent IMs than carcinomas or adjacent NNM (P < .05). Bag-1 mRNA was expressed more in carcinomas from female patients than the male counterparts (P < .05). There was a positive correlation of Bag-1 mRNA expression with invasive depth and venous invasion (P < .05). Our study indicated that aberrant expression and subcellular distribution of Bag-1 might play an important role in the malignant transformation of gastric epithelial cells and should be considered as a biomarker for gastric carcinogenesis, subsequent progression, and prognosis.

Role of fatty acid chain length on the induction of apoptosis by newly synthesized catechin derivatives.

The catechins, a family of polyphenols found in tea, can evoke various responses, including apoptosis. In this study we investigated whether the chemical modification of (-)-epigallocatechin gallate (EGCG) could enhance its apoptosis activity. We found that one of the catechin conjugated with capric acid [(2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl decanoate; catechin-C10] was most potent to induce apoptosis in U937 cells. C10 treatment resulted in a significant increase in reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, cytochrome c release caspase-9 and caspase-3 activation. In addition to this C10 also activated extrinsic pathway significantly as evident by time-dependent increase in Fas expression and caspase-8 activity. C10 mediated cleavage of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Moreover, pre-treatment of cells with anti-oxidant N-acetyl-L-cysteine (NAC) significantly prevented C10-induced apoptosis but did not protect MMP loss. Treatment of cells with pan-caspase inhibitor significantly inhibited apoptosis indicating that caspases are playing key role. In addition to this C10 was found to induce apoptosis in human colon cancer (HCT116) cells while it showed resistance to human keratinocytes (HaCat). In short our results showed that the optimal fatty acid side chain length is required for the apoptosis inducing activity of catechin derivatives in U937 cells.

Novel 3,4-diazabenzotropone compounds (2,3-benzodiazepin-5-ones): synthesis, unique reactivity, and biological evaluation.

Efficient synthesis of 3,4-diazabenzo[b]tropone was first achieved utilizing 4pi-8pi sequential electrocyclic reactions of functionalized benzocyclobutenone derivatives. These compounds are highly electron deficient and readily form amine adducts at ambient temperature. Furthermore, gentle heating resulted in quantitative nitrogen extrusion to produce indenone derivatives. These diazabenzotropones were found to exhibit potent apoptosis-inducing activity against human lymphoma cells. Thus, novel amine-catalyzed nitrogen extrusion reactions and interesting bioactivities were found to be characteristic of these novel diazabenzotropone compounds.

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds.

The combined effects of hyperthermia (44 degrees C, 20 min) or X-rays (10 Gy) and a new class of furan-fused tetracyclic synthesized compounds (DFs), on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (DF1 approximately 6), the combined treatment of 10 microM DF with TIPS (triisopropylsilyloxy) (Designated #3 DF3) and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia but no enhancement was observed if it was combined with X-rays. Enhancement of hyperthermia-induced apoptosis by DF3 in a dose-dependent manner was observed. When the cells were treated first with DF3 at a nontoxic concentration of 20 microM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by DF3. Mitochondrial transmembrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. Externalization of Fas was observed following the combined treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to DF3, and showed subsequent and transient increase in the formation of intracellular hydrogen peroxide (H(2)O(2)), which was further increased when hyperthermia was combined. These results indicate that the intracellular superoxide and H(2)O(2) generated by DF3 enhance the hyperthermia-induced apoptosis via the Fas-mediated mitochondrial caspase-dependent pathway.